Methods for Obtaining Thermostable Enzymes, Dna Polymerase I Variants from Thermus Aquaticus Having New Catalytic Activities, Methods for Obtaining the Same, and Applications to the Same

ABSTRACT

A thermostable mutant DNA polymerase; a method for obtaining the thernmostable mutant DNA polymerase by identifying a thermostable mutant polypeptide exhibiting enzymatic activity, wherein the thermostable mutant polypeptide is a variant of DNA polymerase I obtained from  Thermus aquaticus;  a polynucleotide, an expression vector, and a host cell encoding the thermostable mutant DNA polymerase; and a method of performing a reverse transcription polymerase chain reaction (RT-PCR) utilizing the thermostable mutant DNA polymerase, as well as a kit for facilitating the same.

FIELD OF THE INVENTION

The present invention provides a method for obtaining thermostableenzymes. The present invention also provides variants of DNA polymeraseI from Thermus aquaticus. The present invention further provides methodsof identifying mutant DNA polymerases having enhanced catalyticactivity. The present invention also provides polynucleotides,expression systems, and host cells encoding the mutant DNA polymerases.Still further, the present invention provides a method to carry outreverse transcriptase-polymerase chain reaction (RT-PCR) and kits tofacilitate the same.

DISCUSSION OF THE BACKGROUND

Filamentous phage display is commonly used as a method to establish alink between a protein expressed as a fusion with a phage coat proteinand its corresponding gene located within the phage particle (Marks etal., 1992). The use of filamentous phage particles as a chemical reagentprovides further a strategy to create a complex between an enzyme, itsgene and a substrate (Jestin et al., 1999). This substrate can becross-linked on the surface of filamentous phage using the nucleophilicproperties of coat proteins. If the enzyme is active, conversion of thesubstrate to the product yields a phage particle cross-linked with theproduct, which can be captured by affinity chromatography (Jestin etal., 1999).

Several similar approaches based on product formation for the isolationof genes encoding enzymes using phage display have been described in theliterature for various enzymes (Fastrez et al., 2002). These in vitroselections of proteins for catalytic activity are well suited for usewith large repertoires of about 10⁸ proteins or more. Several librariesof enzyme variants on phage have been constructed and catalyticallyactive proteins with wild type like activities have been isolated(Atwell & Wells, 1999; Heinis et al., 2001; Ponsard et al., 2001; Tinget al., 2001). Mutants with different substrate specificities have beenalso obtained (Xia et al., 2002). In these studies, the fraction ofactive variants in the libraries can be large and it remains unclear howrare an enzyme can be in the initial protein library so as to beselected after iterative selection cycles. Accordingly, there remains acritical need for an efficient process for making and identifyingthermostable enzymes possessing a desired catalytic activity (seediscussion in Vichier-Guerre & Jestin, 2003).

Reverse transcriptases are enzymes that are present generally in certainanimal viruses (i.e., retroviruses), which are used in vitro to makecomplementary DNA (cDNA) from an MRNA template. Practically, reversetranscriptases have engendered significant interest for their use inreverse transcriptase-polymerase chain reaction (RT-PCR). As such, theseproteins lend themselves to be a model system for development of anefficient method of making thennostable enzymes having a desiredactivity.

RNA generally contains secondary structures and complex tertiarysections, accordingly it is highly desired that the RNA be copied in itsentirety by reverse transcription to ensure that integrity of cDNA ismaintained with high accuracy. However, due to the often complicatedsecondary and tertiary structures of RNA, the denaturation temperaturesare generally about 90° C. and, as such, the reverse transcriptase mustbe capable of withstanding these extreme conditions while maintainingcatalytic efficiency.

The classically utilized enzymes for RT-PCR have been isolated from theAMV (Avian myeloblastosis virus) or MMLV (Moloney murine leukemiavirus); however, these enzymes suffer from a critical limitation in thatthey are not thennostable. In fact, the maximum temperature tolerated bymost commercially available reverse transcriptases is about 70° C.

One common approach to overcome this limitation in the existingtechnology with the previously described polymerases has been the use ofa protein chaperones in addition to the polymerase. However, this methodleads to problems associated with environmental compatibility metal ionrequirements, multi-stage procedures, and overall inconvenience.Accordingly, an alternative strategy has been to use thermostablereverse transcriptases. This approach makes it possible to performmultiple denaturation and reverse transcription cycles using only asingle enzyme.

To this end, the DNA-dependent DNA polymerase I of Thermus aquaticus(i.e., Taq polymerase), is thermostable and has reverse transcriptaseactivity only in the presence of manganese. However, when the manganeseion concentration is maintained in the millimolar range the fidelity ofthe enzyme is affected. It has been suggested that the thermostableDNA-dependent DNA polymerase of Bacillus stearothermophilus has reversetranscriptase activity, even in absence of magnesium, but in this caseit is necessary to add a thermostable DNA polymerase for the PCR.

Therefore, there remains a critical need for high efficiency,thermostable enzymes that are capable of catalyzing reversetranscription and subsequent DNA polymerization in “one-pot” RT-PCR.Accordingly, the present invention provides an isolated population ofthermostable reverse transcriptases, which are active in absence ofmanganese, by directed evolution of the Stoffel fragment of the Taqpolymerase.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofidentifying thermostable mutant polypeptides having a catalytic activityby:

-   -   a) packaging a vector in which a gene or fragment thereof        encoding variants of a catalytic domain responsible for the        catalytic activity fused to a gene encoding a phage coat        protein;    -   b) isolation and purification of phage particles;    -   c) heating the phage-mutant polypeptide at a temperature ranging        from 50° C. to 90° C. for a time ranging from less than 1 minute        to several hours;    -   d) cross-linking a specific substrate with a phage particle;    -   e) forming a reaction product from the substrate catalyzed by        the thermostable mutant protein on phage, wherein the        temperature is optionally regulated to be the same or greater or        lower than the temperature of (c);    -   f) selecting the phage particles comprising a variant        nucleotidic sequence encoding for the catalytic domain        responsible for the catalytic activity at the regulated        temperature, by capturing the reaction product or screening for        said reaction product;    -   g) infecting E. coli with the phage particles selected at step        (f);    -   h) incubating the infected E. coli; and    -   i) assessing catalytic activity of the proteins corresponding to        isolated genes.

It is an object of the present invention to provide a thermostablemutant DNA polymerase having at least 80% homology, preferably at least90%, more preferably at least 95%, most preferably at least 97.5%, tothe Stoffel fragment of DNA polymerase I obtained from Thermus aquaticus(residues 13-555 of SEQ ID NO: 26, which correspond to residues 290-832of the wild-type DNA polymerase I from Thermus aquaticus (SEQ ID NO:100)).

To this end, the present invention provides thermostable polypeptideshaving at least 80% homology, preferably at least 90%, more preferablyat least 95%, most preferably at least 97.5%, to residues 13-555 of SEQID NO: 26, wherein said polypeptide has at least one mutation selectedfrom the group consisting of a mutation in amino acids 461-490 of SEQ IDNO: 26 (738 to 767 of the Taq polymerase wild-type sequence SEQ ID NO:100), A331T (position 608 of the Taq polymerase wild-type sequence SEQID NO: 100), S335N (position 612 of the Taq polymerase wild-typesequence SEQ ID NO: 100), M470K (position 747 of the Taq polymerasewild-type sequence SEQ ID NO: 100), M470R (position 747 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), F472Y (position 749 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), M484V (position761 of the Taq polymerase wild-type sequence SEQ ID NO: 100), M484Tposition 761 of the Taq polymerase wild-type sequence SEQ ID NO: 100),and W550R (position 827 of the Taq polymerase wild-type sequence SEQ IDNO: 100), and wherein said polypeptide has improved DNA polymeraseactivity and retains 5′-3′ exonuclease activity. In an object of thepresent invention, the 3′-5′ exonuclease activity of the mutantpolypeptide is inactive.

In an object of the present invention, the thermostable mutant DNApolymerase also has a mutation at one or more position selected fromA331, L332, D333, Y334, and S335 of SEQ ID NO: 26 (positions 608-612 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100).

Therefore, in an object of the present invention, the thermostablemutant DNA polymerase has at least 80% identity to residues 13-335 ofSEQ ID NO: 26 and has a mutation at one or more position selected fromH203, F205, T232, E253, Q257, D274, L275, I276, V309, I322, A331, L332,D333, Y334, S335, I361, R374, A384, T387, Y419, P493, M498, G499, M502,L503, V506, R518, A523, A526, P539, E543, and W550 of SEQ ID NO: 26. Thepresent invention also embraces polynucleotides encoding the same.

The present invention also provides thermostable polypeptides having atleast 80% homology, preferably at least 90%, more preferably at least95%, most preferably at least 97.5%, to residues 13-335 of SEQ ID NO:26, wherein said polypeptide has at least one mutation selected from thegroup consisting of H203R (position 480 of the Taq polymerase wild-typesequence SEQ ID NO: 100), F205L (position 482 of the Taq polymerasewild-type sequence SEQ ID NO: 100), T232S (position 509 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), E253G (position 530 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), Q257R (position534 of the Taq polymerase wild-type sequence SEQ ID NO: 100), D274G(position 551 of the Taq polymerase wild-type sequence SEQ ID NO: 100),L275H (position 552 of the Taq polymerase wild-type sequence SEQ ID NO:100), L275P (position 552 of the Taq polymerase wild-type sequence SEQID NO: 100), I276F (position 553 of the Taq polymerase wild-typesequence SEQ ID NO: 100), V309I (position 586 of the Taq polymerasewild-type sequence SEQ ID NO: 100), I322N (position 599 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), A331V (position 608 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), S335N (position612 of the Taq polymerase wild-type sequence SEQ ID NO: 100), I361F(position 638 of the Taq polymerase wild-type sequence SEQ ID NO: 100),R374Q (position 651 of the Taq polymerase wild-type sequence SEQ ID NO:100), A384T (position 661 of the Taq polymerase wild-type sequence SEQID NO: 100), T387A (position 664 of the Taq polymerase wild-typesequence SEQ ID NO: 100), Y419C (position 696 of the Taq polymerasewild-type sequence SEQ ID NO: 100), Y419N position 696 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), E465K (position 742 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), M470K (position747 of the Taq polymerase wild-type sequence SEQ ID NO: 100), M470R(position 747 of the Taq polymerase wild-type sequence SEQ ID NO: 100),F472Y (position 749 of the Taq polymerase wild-type sequence SEQ ID NO:100), F472S (position 749 of the Taq polymerase wild-type sequence SEQID NO: 100), A487T (position 764 of the Taq polymerase wild-typesequence SEQ ID NO: 100), IK490E (position 767 of the Taq polymerasewild-type sequence SEQ ID NO: 100), P493T (position 770 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), M498T (position 775 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), G499E (position776 of the Taq polymerase wild-type sequence SEQ ID NO: 100), M502K(position 779 of the Taq polymerase wild-type sequence SEQ ID NO: 100),L503P (position 780 of the Taq polymerase wild-type sequence SEQ ID NO:100), V506I (position 783 of the Taq polymerase wild-type sequence SEQID NO: 100), A523V (position 800 of the Taq polymerase wild-typesequence SEQ ID NO: 100), A526V (position 803 of the Taq polymerasewild-type sequence SEQ ID NO: 100), P539S (position 816 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), E543K (position 820 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), and W550R(position 827 of the Taq polymerase wild-type sequence SEQ ID NO: 100),and wherein said polypeptide has improved DNA polymerase activity andretains 5′-3′ exonuclease activity. In an object of the presentinvention, the 3′-5′ exonuclease activity of the mutant polypeptide isinactive.

In a particular object of the present invention, the mutant DNApolyrnerase has a sequence corresponding to residues 13-335 of one ofthe following sequences: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, and SEQ ID NO: 38.

In another object of the present invention, the mutant DNA polymerasehas a sequence corresponding to residues 1-543 of one of the followingsequences: SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ IDNO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, andSEQ ID NO: 99. Further, in another object of the present invention arepolynucleotides that encode for the aforementioned thermnostable mutantDNA polymerases.

In yet another object of the present invention is a kit for DNAamplification, which contains: (a) one or more of the aforementionedthermostable mutant DNA polymerases; (b) a concentrated buffer solution,wherein when said concentrated buffer is admixed with the isolatedpolypeptide the overall buffer concentration is 1X; (c) one or moredivalent metal ion (e.g., Mg²⁺ or Mn²⁺); and (d) deoxyribonucleotides.

In yet another object of the present invention is a method of reversetranscribing RNA by utilizing the inventive thermostable mutant DNApolymerases.

In still a further object of the present invention is a phage-displaymethod for identifying thermostable mutant DNA polymerases in which theStoffel fragment has been mutated, while the DNA polymerase activity and5′-3′ exonuclease activity has been maintained and/or enhanced.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following Figures in conjunction with thedetailed description below. In the following legends, polymerase ecorresponds to SEQ ID NO: 26 containing a R518G mutation (see Examples).

FIG. 1 shows the reverse transcriptase activity of phage-polymerasesassessed as obtained after different rounds of selection in the presenceof Mg²⁺ or Mn²⁺ ions. The lane labels correspond to the following:

MnCl2 MgCl₂ a: phage-polymerases of round 6 h: phage-polymerases ofround 6 b: phage-polymerases of round 5 i: phage-polymerases of round 5c: phage-polymerases of round 4 j: phage-polymerases of round 4 d:phage-polymerases of round 3 k: phage-polymerases of round 3 e:phage-polymerases of round 2 l: phage-polymerases of round 2 f:phage-polymerases of round 1 m: phage-polymerases of round 1 g:phage-polymerases of initial n: phage-polymerases of initial populationpopulation

FIG. 2 shows the reverse transcriptase activity of phage-polymerasesassessed as obtained after different rounds of selection in the presenceof Mg²⁺ ions. The lane designations in FIG. 2 are as follows:

Phage-polymerase preheated at 65° C. for 5 min. Phage-polymerase notpreheated a: phage-polymerases of initial h: phage-polymerases ofinitial population population b: phage-polymerases of round 1 i:phage-polymerases of round 1 c: phage-polymerases of round 2 j:phage-polymerases of round 2 d: phage-polymerases of round 3 k:phage-polymerases of round 3 e: phage-polymerases of round 4 1:phage-polymerases of round 4 f: phage-polymerases of round 5 m:phage-polymerases of round 5 g: phage-polymerases of round 6 n:phage-polymerases of round 6 o: control AMV-RT, 1 U p: control AMV-RT,0.1 U q: control AMV-RT, 0.01 U r: control AMV-RT, 0.001 U

FIG. 3 shows the reverse transcriptase activity of various monoclonalphage-polymerases obtained after round 6 in the presence of Mg²⁺ ions.The lane designations in FIG. 3 are as follows: s=SEQ ID NO: 38; a=SEQID NO: 20; d=SEQ ID NO: 24; g=SEQ ID NO: 28; C=AMV-RT; i=SEQ ID NO: 30;m=SEQ ID NO: 32; n=SEQ ID NO: 34; b=SEQ ID NO: 22; and q=SEQ ID NO: NO:36.

FIG. 4 shows the reverse transcriptase activities and the polymeraseactivities of monoclonal phage-polymerases obtained after the round 6 inthe presence of Mg²⁺ or Mn²⁺ ions. The lane designations in FIG. 4 areas follows: a=SEQ ID NO: 20; b=SEQ ID NO: 22; d=SEQ ID NO: 24; and e=SEQID NO: 26 (containing an R518G mutation).

FIG. 5 shows purified mutant polymerases a, b, and d used in polymerasechain reaction. The lanes in the gel appearing in FIG. 5 include thetlhree clones corresponding to clones a, b and d on FIG. 4. In addition,the positive control was performed using the Stoffel fragment polymerasee and the polymerase AMV-RT (Promega). The lanes in FIG. 5 are asfollows:

-   -   lane 1: molecular weight marker: PhiX phage DNA digested by        HaeIII    -   lane 2: control AMV-RT    -   lane 3: b=SEQ ID NO: 22    -   lane 4: a=SEQ ID NO: 20    -   lane 5: e=SEQ ID NO: 26 (containing an R518G mutation)    -   lane 6: d=SEQ ID NO: 24

FIG. 6 shows the purification control of proteins after Co⁺² affinitychromatography.

Variant a

-   -   Well 1=8 μl Fraction 1    -   Well 2=8 μl Fraction 2    -   Well 3=8 μl Fraction 3    -   Well 4=8 μl Fractions 1+2+3 pooled and concentrated on Ultra        Amicon 4 column (Millipore).    -   OD (a)=0.869 mg/ml.

Variant d

-   -   Well 1=8 μl Fraction 1    -   Well 2=8 μl Fraction 2    -   Well 3=8 μl Fraction 3    -   Well 4=8 μl Fractions 1+2+3 pooled and concentrated on Ultra        Amicon 4 column (Millipore).    -   OD (d)=0.908 mg/ml.

Variant e

-   -   Well 1=8 μl Fraction 1    -   Well 2=8 μl Fraction 2    -   Well 3=8 μl Fraction 3    -   Well 4=8 μl Fractions 1+2+3 pooled and concentrated on Ultra        Amicon 4 column (Millipore).    -   OD (e)=0.958 mg/ml.

Variant b

-   -   Well 1=8 μl Fraction 1    -   Well 2=8 μl Fraction 2    -   Well 3=8 μl Fraction 3    -   Well 4=8 μl Fractions 1+2+3 pooled and concentrated on Ultra        Amicon 4 column (Millipore).    -   OD (b)=0.514 mg/ml.

M is the low range SDS PAGE molecular weight standards. Low Range(BIO-RAD, Ref. 161-0304). Bands located at 97.4 kDa; 66.2 kDa; 45 kDa;31 kDa; 21 kDa and 14.4 kDa.

FIG. 7 shows protein e purification by Co²⁺ affinity chromatographyfollowed by heparin affinity chromatography.

Lanes 1 and 2 correspond to the protein e after purification by affinitychromatography on a Co²⁺ colunmn. Lanes t1, t2, t3, t4 coxresponds tothe most concentrated fractions after the two-step purification byaffinity chromatography, first on a Co²⁺ column and second on a heparincolumn. M is the low range SDS PAGE molecular weight standards (Biorad).

FIG. 8 shows 20% Polyacrylamide gels electrophoresis obtained withvariants e, a, d and b for the test of primer extension.

FIG. 9 shows products of PCR with polymerase e and variant a as shown byagarose gel after electrophoresis. M is the marker Smartladder(Eurogentec).

FIG. 10 shows the results of a PCR reaction using variant a. Resultsshown on a 2% agarose gel: deposit of 15 μl of amplification product.

Product of amplification=475 bp.

-   -   1. SmartLadder 100 bp (EUROGENTEC, Ref MW-1800-02, 200 lanes,        Smallfragment)    -   2. Variant a MJ Research    -   3. Variant a/MJ Research    -   4. SmartLadder 100 bp    -   5. Variant a/Applied BioSystems    -   6. Variant a/Applied BioSystems

FIG. 11 shows Product of RT PCR “one pot” with variant a. As shown inthe agarose gel after electrophoresis, M is the marker of phage PhiX DNAdigested by the restriction enzyme HaeIII.

FIG. 12 shows the reverse transcriptase activity of various monoclonalphage-polymerases obtained after round 6 in the presence of Mg²⁺ ions.The lane designations in FIG. 12 are as follows: rt1=SEQ ID NO: 63;rt2=SEQ ID NO: 65; rt3=SEQ ID NO: 67; rt16=SEQ ID NO: 69; rt18=SEQ IDNO: 71; rt25=SEQ ID NO: 73; rt26=SEQ ID NO: 75; rt28=SEQ ID NO: 77;rt30=SEQ ID NO: 79; rt31=SEQ ID NO: 81; rt33=SEQ ID NO: 83; rt36=SEQ IDNO: 85; rt43=SEQ ID NO: 87; rt59=SEQ ID NO: 89; rt64=SEQ ID NO: 91;rt70=SEQ ID NO: 93; rt78=SEQ ID NO: 95; rt80=SEQ ID NO: 97; rt86=SEQ IDNO: 99; and nd=not described here.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by a skilled artisanin enzymology, biochemistry, cellular biology, molecular biology, andthe medical sciences.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The present invention provides a method of identifying thermostablemutant polypeptides having a catalytic activity comprising:

-   -   a) packaging a vector in which a gene or fragment thereof        encoding variants of a catalytic domain responsible for the        catalytic activity fused to a gene encoding a phage coat        protein;    -   b) isolation and purification of phage particles;    -   c) heating the phage-mutant polypeptide at a temperature ranging        from 50° C. to 90° C., preferably from 55° C. to 65° C., more        preferably at 65° C. for a time ranging from 30 seconds to        several hours, preferably from 1 minute to 3 hours, more        preferably from 5 minutes to 2 hours, most preferably 10 minutes        to 1 hour;    -   d) cross-linking a specific substrate with a phage particle;    -   e) forming a reaction product from the substrate catalyzed by        the thermostable mutant polypeptide on phage, wherein the        temperature is optionally regulated to be the same or greater or        lower than the temperature of (c) (i.e., from 25° C. to 70° C.,        preferably from 37° C. to 70° C. and more preferably at 65° C.);    -   f) selecting the phage particles comprising a variant        nucleotidic sequence encoding for the catalytic domain        responsible for the catalytic activity at the regulated        temperature, by capturing the reaction product or screening for        said reaction product;    -   g) infecting E. coli with phage particles selected at (f);    -   h) incubating the infected E. coli; and    -   i) assessing catalytic activity of the proteins corresponding to        isolated genes.

In the embodiment above, the gene or fragment thereof encoding variantsof a catalytic domain may be directly or indirectly fused to the geneencoding a phage coat protein. When the gene or fragment thereofencoding variants of a catalytic domain and the gene encoding a phagecoat protein are indirectly fused it is preferred that the fusion bethrough a peptide or polypeptide linker.

Within this above-recited embodiment, steps (a) to (h) may be repeated 0to 20 times, preferably 1 to 15 times, more preferably 2 to 10 times,most preferably 3 to 7 times.

The method comprising a single cycle (repeated 0 times) is particularlyadapted to high throughput screening, when steps are repeated from 3 to7 times, the method is better adapted for classical empirical screening.

The peptide utilized within this embodiment is selected from the groupconsisting of: a flexible linker such as a glycine rich linker such as(SG₄)n or the sequence SG₄CG₄ (residues 3-12 of SEQ ID NO: 39),

-   -   human calmodulin (SEQ ID NO: 46, the DNA encoding SEQ ID NO: 46        is SEQ ID NO: 56), and    -   hexahistidine binding single chain variable fragment (Gritter        M.G., 2002) consisting of:        -   (i) Anti-His Tag Antibody 3D5 Variable Heavy Chain (SEQ ID            NO: 47)        -   (ii) Linker (SEQ ID NO: 48)        -   (iii) Anti-His Tag Antibody 3D5 Variable Light Chain (SEQ ID            NO: 49).

Moreover, the polypeptide linker is selected from the group consistingof: any protein binding the substrate at high temperature, any catalyticdomain such as 5′ to 3′ exonuclease (from Thermus thermophilus, SEQ IDNO: 50), or 3′ to 5′ exonuclease (from E. coli, SEQ ID NO: 51),Catalytic domain of Bacillus circulans cyclodextringlycosyltransferase(SEQ ID NO: 52 the DNA is in SEQ ID NO: 57), Catalytic domain ofBordetella pertussis adenylate cyclase (SEQ ID NO: 53—the DNA is in SEQID NO: 58), Bacillus amyloliquefaciens serine protease subtilisin (SEQID NO: 54—the DNA is in SEQ ID NO: 59), and Catalytic domain of Bacillussubtilis lipase A (SEQ ID NO: 55, Quax W. J., 2003).

As used in the present invention, the cross-linking between the specificsubstrate of the catalytic domain of the polypeptide with the phageparticle is made by a cross-linking agent selected from the groupconsisting of a: maleimidyl group, iodoacetyl group, disulfidederivative and any other thermostable link (conducting to a stableprotein-protein interaction or protein-molecule interaction).

In a preferred embodiment, the catalytic domain may be the catalyticdomain of an enzyme selected from the group consisting of: a polymerase,an alpha-amylase (substrate such as starch), a lipase (substrate such asester), a protease (modified or not modified peptide or polypeptide assubstrate), a cyclodextringlycosyltransferase, and an adenylate cyclase.

In another embodiment, the assessment of the catalytic activity of step(f) is made by means of a DNA polymerization.

In yet another embodiment of the present invention, step (b) may beperformed after (e) of cross-linking or during (h) of assesing catalyticactivity.

As a general method for the isolation of thermostable enzymes and theirgenes the following should be noted:

First, the gene encoding variants of a catalytic domain are fused to thegene gencoding a phage coat protein (such as filamentous phage g3, g6,g7, g9 or g8 protein or of other phage/virus particles) either directlyor using a peptide or polypeptide linker such as a short peptidesequence or a protein or a protein domain. These genes encoding phagecoat proteins may be fused either at the 3′ or at the 5′ terminusdepending on whether the N- or the C-termini of the proteins are locatedon the outside of the particle.

This is done either using a phage vector or a phagemid vector used witha helper phage.

Second, the phage-variant enzymes may be heated at a preferredtemperature of 65° C. for 1 minute or for several hours as appropriate.This step can be performed before or after the substrate cross-linking(maleimidyl group derivatised substrate (DNA primer) crosslinked to thephage particle) and catalysis (DNA polymerisation) steps. Catalysis ispreferably at 65° C. for 2 minutes, but can be done at any temperaturebetween 0° C. and 100° C. Crosslinking is typically performed for 2hours at 37° C., but can be done at other temperatures (highertemperature may increase maleimidyl hydrolysis versus maleimidyl phagecross-linking).

It is worth noting that the link between the gene and the correspondingenzyme variants is unaltered by high temperatures and the phage particleare still infective and the genes selected can be amplified by E. coliafter infection (cf. for example, Kristensen P, Winter G., 1998).

By way of example of the aforementioned embodiments, the presentinvention relates to a purified, thermostable DNA polymerase purifiedfrom Thermus aquaticus and recombinant means for producing the enzyme.Thermostable DNA polymerases are useful in many recombinant DNAtechniques, especially nucleic acid amplification by the polymerasechain reaction (PCR).

Directed protein-evolution strategies generally make use of a linkbetween a protein and the encoding DNA. In phage-display technology,this link is provided by fusion of the protein with a coat-protein thatis incorporated into the phage particle containing the DNA. Optimizationof this link can be achieved by adjusting the signal sequence of thefusion.

Linking of a gene to its corresponding polypeptide is a central step indirected protein evolution toward new functions. Filamentousbacteriophage particles have been extensively used to establish thislinkage between a gene of interest and its protein expressed as a fusionproduct with a phage coat protein for incorporation into the phageparticle. Libraries of proteins displayed on phage can be subjected toin vitro selection to isolate proteins with desired properties togetherwith their genes.

Creating a link between a gene and a single corresponding protein wasachieved by making use of a phagemid for expression of the fusionprotein and of a helper phage for assembly of the phage particles. Thisapproach, yielding a monovalent display of protein, was found to beessential to avoid avidity effects or chelate effects, which introducestrong biases during in vitro selections for affinity. However, it alsoproduces phage particles that do not display any protein of interest andwhich thereby represent a background in evolution experiments.

To optimize the link between a gene and a single corresponding protein,several methods have been used. For example, the periplasmic factor Skpwas found to improve the display of single-chain Fv antibodies onfilamentous phage (Bothmann, H. and Pluckthun, A., 1998). In a previousstudy, the present inventors showed that specific signal sequences foroptimal display on phage of the Taq DNA polymerase I Stoffel fragmentcan be isolated from a library of more than 10⁷ signal sequences derivedfrom pelB (Jestin, J. L., Volioti, G. and Winter, G., 2001). Signalsequences, once translated, are recognized by the bacterial proteinexport machinery. The polypeptide is then exported in the bacterialperiplasm before cleavage of the signal peptide by the signal peptidase,thereby releasing the mature protein.

A short sequence, m (SG₄CG₄; residues 3-12 of SEQ ID NO: 39), at theC-terminus of the signal sequence, was initially introduced as apotential cross-linking site of substrates on phage that may be usefulfor selections by catalytic activity. This glycine-rich sequence mayalso be important for preventing structure formation at the peptidasecleavage site or for defining two independently folding units in thepre-protein. The glycine-rich sequence may then improve the signalsequence processing and finally lead to a greater ratio of proteinfusions on phage. The present inventors, therefore, evaluated the effectof a selected signal sequence on the display of proteins on phage, aswell as the effect of the m sequence at the C-terminus of the signalpeptide.

In an embodiment of the present invention is a method of identifyingthermostable mutant polymerases derived from the Stoffel fragment of Taqcomprising:

-   -   a) packaging a vector in which a polynucleotide encoding a phage        coat protein is fused to a polynucleotide encoding a protein        having at least 80% identity to residues 13-555 of SEQ ID NO: 26        into a phage;    -   b) expressing the fusion protein;    -   c) isolation (selection) of phage particles;    -   d) infecting E. coli and incubating the infected E. coli;    -   e) detecting the fusion protein;    -   f) assessing polymerase activity.

In this method, evolutionarily advantageous mutants may be identified byrepeating steps (b)-(f) 0 to 25 times, preferably 0-20 times, morepreferably 1-15 times, a most preferably 2 to 10 times. The methodcomprising one cycle (repeated 0 times) is particularly adapted to highthroughput screening, when steps are repeated from 3 to 7 times, themethod is better adapted for classical emprirical screening.

In a preferred embodiment, the phage coat protein is fused to saidpolynucleotide encoding a protein having at least 80% identity toresidues 13-555 of SEQ ID NO: 26 by way of a linker having a sequencerepresented by residues 3-12 of SEQ ID 39.

By way of example, Applicants provide the following exemplary discussionof the phage-display method of the present invention and refer toStrobel et al., 2003, which is incorporated herein by reference in itsentirety:

The amino acid signal sequences are that may be attached to theN-terminus of the proteins of the present invention:

pe1B: MKYLLPTAAAGLLLLAAQPAMA; (SEQ ID NO: 41) 17:MKTLLAMVLVGLLLLPPGPSMA; (SEQ ID NO: 42) I10: MRGLLAMLVAGLLLLPIAPAMA;(SEQ ID NO: 43) and I12: MRRLLVIAAVGLLLLLAPPTMA. (SEQ ID NO: 44)

The present inventors goal was to increase the display of proteins atthe surface of filamentous phages. As model proteins, the presentinventors chose the catalytic domains of adenylate cyclases from E. coli(ACE) and from B. pertussis (ACB). The present inventors also examinedthe display of two different enzymes, an adenylate cyclase and theStoffel fragment of Taq DNA polymerase I, incorporated into phageparticles as single polypeptide fusion products with minor coat proteinp3. In this work, the present inventors evaluated the effects of twosignal peptides (pelB and 17) and of the short peptide (m; residues 3-12of SEQ ID NO: 39) at the N-terminus of the fusion of these enzymes withp3. One other construct, deriving from the selected signal peptide 112,is also mentioned here, and the data are summarized together withpreviously published data for the selected signal sequences 110 and 112(Jestin et al., 2001).

The phage particles were produced by using a helper phage, KM13(Kristensen et al., 1998), for assembly of the particles, and by usingphagemids pHEN1 (Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., etal., 1991), pHEN117, and pHEN1112 (Jestin et al., 2001) encoding the p3fusion proteins. These phagemid vectors differ in their signal sequence:pelB is from Erwinia caratovora pectate lyase B (Lei, S. P., Lin, H. C.,Wang, S. S., Callaway, J., et al., 1987), whereas signal sequences 17,110, and 112, were selected from a library of more than 10⁷ signalsequences for optimal display of the Stoffel fragment on filamentousphage (Jestin et al., 2001). For all 17 phagemids encoding the differentfusion proteins described in this work, the present inventors observedstandard titers of infective particles, which were all in the range of1.4×10¹⁰-7.8×10¹⁰ phages/mL of culture medium. Furthennore, enzymaticactivities were detected for all phage-cyclase particles by thin layerchromatography and by HPLC (data not shown).

The efficiency of protein display on phage was evaluated through twoapproaches. The first makes use of the engineered helper phage KM13(Kristensen et al., 1998) to measure the fraction of infective phageparticles that display a fusion product. The p3 fusion protein providedby the phagemid and tlhe p3 protein provided by the helper phage competefor incorporation into the phage particles. The helper phage p3 isengineered so as to contain a protease cleavage site between domains 2and 3 of p3. In phage particles that contain only helper p3 copies, nofull p3 copy is available for bacterial infection after proteasetreatment: the phage particles are noninfective. If a phage particle hasincorporated a p3 fusion protein, one copy of the three-p3 domainsremains after protease cleavage, and is sufficient for infection of E.coli. The trypsin-resistant fraction of phage is therefore a measure ofprotein display on infective phages. With this method, the display offusion proteins was found to vary over more than two orders of magnitudefor each cyclase, depending on the signal sequence and on neighboringsequences. Among the phagemid vectors containing the selected signalsequence 17, three of the four fusion proteins that the presentinventors studied (AC-p3 and AC-Stoffel-p3, where AC is the adenylatecyclase catalytic domain of E. coli or B. pertussis) were remarkablywell incorporated into phage particles: more than one phage particle outof ten displayed an enzyme. No more than one particle in 300 displayedthe E. coli cyclase fused to the Stoffel fragment and to protein 3, andbetter display of this protein could not be found among the constructstested.

The peptide m, SG₄CG₄, at the N-terminus of the mature fusion protein,was found to increase the display of B. pertussis cyclase-polymerasefusion on phage, by 100-fold for signal sequence 17 and by 10-fold forpelb. For this fusion, the worst display ratios are significantlyimproved with peptide m. Display of B. pertussis cyclase on phage washigh in all cases, such that a marginal improvement due to the m peptidewas found for signal sequence 17, and improvement within the limits ofexperimental error for pelb. Concerning the E. coli cyclase protein,peptide m decreases the latter's display by a factor of 30 to 40. Forthe E. coli cyclase-polymerase fusion, peptide m showed no significanteffect with the signal sequence pelb and a small improvement with signalsequence 17.

Significant effects of the signal sequence on phage display weredetected for three of the four fusions in the present inventors' study:from 5- to about 20-fold improvements in display on phage were noted forsubstitution of pelB by signal sequence 17. In the case of the B.pertussis cyclase-p3 fusion protein, incorporation of the fusion proteininto phage particles was high, whether the signal sequence was pelB, 17,or 112. Indeed, for the selected signal sequence 112, up to 40% ofinfective phage particles displayed an enzyme at the surface offilamentous phage.

When two enzymes were simultaneously displayed on phage (either E. colior B. pertussis adenylate cyclase and the Stoffel fragment polymerase),the present inventors noted that the incorporation of p3 fusion productswas significantly reduced in most cases. Remarkably, about half of theinfective phage particle& displayed a B. pertussis adenylatecyclase-Stoffel fragment polymerase-p3 protein fiusion when the selectedsignal sequence 17 and the short N-terminal peptide m were present inthe construct.

The second approach to estimating the level of fusion proteinsincorporated into phage particles relies on the detection of p3 domain 3by a morioclonal antibody (Tesar, M., Beckmann, C., Rottgen, P., et al.,1995) after SDS-PAGE and Western blotting of denatured phage particles.These results are in accordance with the data the present inventorsobtained by measuring the trypsin-resistant fraction of infectivephages. All fusion products expressed on phage and which correspond to atrypsin-resistant fraction of phage higher than 0.1 are indeed observedby Western blot analysis.

The present inventors aim to direct the evolution of adenylate cyclasesby in vitro selection using a chemistry involving filamentous phage.This should provide a tool for the engineering of adenylate cyclases aswell as a strategy for the functional cloning of this class of enzymes.Recent in vitro selection methods for catalytic activity using phagedisplay have been designed as affinity chromatography methods for thereaction product linked to the phageenzyme that catalyzed the reactionfrom substrate to product. These selection methods were established withenzymes such as nuclease (Pedersen, H., H6lder, S., Sutherlin, D. P., etal., 1998), DNA polymerase (Jestin et al., 1999), peptidase (Dematris,S., Huber, A., et al., 1999; Heinis, C., Huber, A. et al., 2001),peptide ligase (Atwell et al., 1999), and beta-lactamase (Ponsard etal., 2001). They require an efficient display of enzyme on phage and amethod to link the substrate/product to phage-enzymes.

In the work reported here, the present inventors investigated thedisplay of adenylate cyclases from B. pertussis and from E. coli onfilamentous phage, and the display of two independent enzymes, anadenylate cyclase and the Taq DNA polymerase I Stoffel fragment. TheStoffel fragment (Lawyer, F. C., Stoffel, S., Saiki, R. K., et al.,1989) could be used as a tool to establish an in vitro selection forcyclase activity as follows: the polymerase domain may serve as ananchor of the substrate ATP on phage through double-stranded DNA used asa linker with a high affinity for the fusion protein. Another approachto cross-linking substrate and phage involves introduction of the thiolgroup of a cysteine residue within peptide m (SG₄CG₄), at the N-terminusof the mature fusion protein and at the C-terminus of the fusionprotein's signal sequence (Jestin et al., 1999).

The signal sequences 17, 110, and 112, used in the present inventors'study had been selected from large libraries of pelB mutants for optimaldisplay of the Stoffel fragment-p3 protein fused to the peptide mn(Jestin et al., 2001). It was therefore important to further investigatewhich sequence context was essential for selection of these signalsequences, either the short peptide in or the entire gene.Interestingly, the present inventors found that the presence or theabsence of this short peptide, SG₄CG₄, can yield up to 100-foldincreases in the display of a fusion protein on filamentous phage. Thisstrong effect was observed for the B. pertussis cyclase-Stoffel-p3fusion as well as for the E. coli cyclase-p3 fusion in the case of thesignal sequence 17 (Table 2). Of further note is that the signalsequences 17 and 112, yield generally better levels of protein displayon phage than does pelB (FIG. 3). This improved display of proteinsmight be ascribed to the different targeting modes of the signalsequences. These selected signal sequences that improve the display ofproteins on phage should therefore be useful in other systems.

Our study highlights the important effects of the signal sequence and ofa short peptide at the C-terminus of the signal sequence on the displayof proteins on phage. Apart from the previously stated conclusions thatthe selected signal sequence 17 often yields an improved display ascompared with pelB, and that sequence m can have drastic effects on thelevel of protein display, the set of protein fusions described here isnot sufficient to define any further rules about sequences and optimaldisplay of proteins on phage. Indeed, incorporation of a fusion proteininto a phage particle is the result of a complex sequence of eventsinvolving fusion gene transcription and translation, folding, and exportof the fusion protein, as well as cleavage of the signal sequence.

Two approaches, however, can be envisaged for efficient display ofproteins on bacteriophage. First, directed signal peptide evolutionexperiments can be undertaken for any defined protein so as to isolate asignal sequence for optimal display on phage. This approach wasdescribed previously in the case of the Stoffel fragment of Taq DNApolymerase I (Jestin et al., 2001). A more straightforward and quickerapproach consists of the screening of several phagemid vectors thatdiffer in their signal sequences and, more generally, in theirregulatory sequences. In this report the present inventors have shownthat for three of the four fusion proteins tested, excellent cyclasedisplay levels can be obtained: more than one phage in ten displays anenzyme. Such display levels for large proteins should be useful forfurther approaches to directed protein evolution.

With use of the phagemid strategy, almost every particle expresses a p3copy provided by the phagemid if no gene fusion has been engineered orif the insert from the gene fusion has been deleted. On the contrary,about one phage particle in a thousand incorporates large fusionproteins such as cyclase-Stoffel fragment-p3 fusions. This indicatesthat for an equal mixture of two genes, thousand-fold differences inexpression of the corresponding proteins on phage particles can beobtained. This bias may be of no importance if enrichment factors perselection round are much larger than 10³, but it may otherwisesignificantly alter the outcome of evolution experiments. Similarprotein expression levels on phage of different genes would be useful tominimize biases introduced by successive amplifications in evolutionexperiments. The use of sets of phagemid vectors that differ by theirsignal sequences and by neighboring sequences might be of interest forbetter representation of protein libraries on filamentous phage.Additionally, the display of two distinct enzymes on single phageparticles might be useful to direct their coevolution, especially in thecase of two enzymes involved in the same metabolic pathway with anunstable reaction intermediate.

By insertion or by deletion of the short peptide sequence SG₄CG₄ (m;residues 3-12 of SEQ ID NO: 39) at the C-terminus of the signal sequence(i.e., immediately upstream (N-terminal) to the variant Stoffelfragments of the present invention), the present inventors have shownthat two enzymes can be very efficiently expressed as singlepolypeptides on the surface of filamentous bacteriophage by using thephagemid strategy. The model proteins described in this study are thecatalytic domains of adenylate cyclases of B. pertussis or of E. coli ,fused or not fused to the Stoffel-fragment DNA polymerase.

On average, the present inventors found the best display levels for theselected signal sequence 17, which had been previously selected from alarge library for optimal display on phage of the Stoffel fragment, andnot for the commonly used signal sequence pelB. Yet the presentinventors observed striking differences in display levels of theseenzymes on the surfaces of phage particles, depending on the shortN-terminal peptide m. The findings reported here should be useful forthe display of large and of cytoplasmic proteins on filamentous phageparticles, and more generally for protein engineering using phagedisplay.

It is important to note that within the present application the terms“protein,” “polymerase,” “enzyme,” “clone,” and “variant” are consideredto be equivalent terms when used to qualify, name, or otherwisedesignate the mutant Stoffel fragments of the present invention.Further, in the context of the present invention the term “Stoffelfragment,” “the Stoffel fragment of DNA polymerase I obtained fromThermus aquaticus” or similar terms are used herein, and is frequentlyassociated with SEQ ID NO: 26. Also in the present invention variant eis used in conjunction with SEQ ID NO: 26, as this sequence correspondsto the native Stoffel fragment of DNA polymerase I obtained from Thermusaquaticus, but contains a R518G (in the context of the full-length Taqsequence, this is a R795G mutation). It is to be understood thatreference to variant e is sometimes used as a short hand for residues13-555 of SEQ ID NO: 26, wherein SEQ ID NO: 26 actually corresponds tothe native Stoffel fragment of DNA polymerase I obtained from Thermusaquaticus.

Residues 1-12 of SEQ ID NO: 26 correspond to SEQ ID NO: 39, whichcontain 2 residues from the signal sequence (MetAla) and 10 residues(SerGly₄CysGly₄) corresponding to a linker that had been introduced atN-terminus of the mature fusion protein on phage so as to introduce acysteine residue that might be important for substrate cross-linking onphage. Further residues 556-562 correspond to residues 1-7 of SEQ ID NO:40, which is the resultant sequence following thrombin cleavage of thesequence of SEQ ID NO: 40. It should also be understoo d that thepresent invention embraces sequences corresponding to residues 13-555 ofSEQ ID NO: 26 as defined herein, as well as sequences in which theN-terminus and C-terminus contain signal sequences, linker sequences,purification tags, and/or fusion constructs.

The term “thermostable” enzyme refers to an enzyme that is stable over atemperature range of approximately 55° C. to 105° C. In particular,thermostable enzymes in accordance with the present invention are heatresistant and catalyze the template directed DNA synthesis. Preferably,the activity of the thermostable enzymes of the present is at least 50%of activity, preferably at least 75%, more preferably at least 85%, ofthe wild-type enzyme activity over the same temperature range. In aparticularly preferred embodiment, the thermostable enzyme of thepresent invention exhibits at least 50% of activity, preferably at least75%, more preferably at least 85%, of the wild-type enzyme activity whensaid wild-type enzyme activity is measured under optimal conditions.Moreover, it is preferable that the “thermostable” enzyme does notbecome irreversibly denatured when subjected to the elevatedtemperatures and incubation time for denaturation of double-strandednucleic acids, as well as the repetitive cycling between denaturation,annealing, and extension inherent to PCR-based techniques.

As used herein, the term “reduced” or “inhibited” means decreasing theactivity of one or more enzymes either directly or indirectly. Thedefinition of these terms also includes the reduction of the in vitroactivity, either directly or indirectly, of one or more enzymes.

The term “enhanced” as used herein means increasing the activity orconcentration one or more polypeptides, which are encoded by thecorresponding DNA. Enhancement can be achieved with the aid of variousmanipulations of the bacterial cell, including mutation of the protein,replacement of the expression regulatory sequence, etc.

In order to achieve enhancement, particularly over-expression, thenumber of copies of the corresponding gene can be increased, a strongpromoter can be “operably linked,” or the promoter- and regulationregion or the ribosome binding site which is situated upstream of thestructural gene can be mutated. In this regard, the term “operablylinked” refers to the positioning of the coding sequence such that apromoter, regulator, and/or control sequence will function to direct theexpression of the protein encoded by the coding sequence locateddownstream therefrom.

Expression cassettes that are incorporated upstream of the structuralgene act in the same manner. In addition, it is possible to increaseexpression by employing inducible promoters. A gene can also be usedwhich encodes a corresponding enzyme with a high activity. Expressioncan also be improved by measures for extending the life of the mRNA.Furthermore, preventing the degradation of the enzyme increases activityas a whole. Moreover, these measures can optionally be combined in anydesired manner. The definition of these terms also includes theenhancement of the in vitro activity, either directly or indirectly, ofone or more enzymes.

It is to be understood that the in addition to the following variantStoffel polypeptides and polynucleotides encoding the same, the presentinvention also embraces full-length Taq polymerase enzymes in which thespecifically identified mutations have been effectuated. The presentinvention further embraces full-length polymerases beyond those of thegenus Thermus in which the domain equivalent in function to the Stoffelfragment is replaced by the variant sequences of the present inventionthereby imparted or enhancing thermostability of the resultantpolymerase. The skilled artisan would readily appreciate methods ofmutagenesis and/or sub-cloning to alter the sequence of the Taqpolymerase to incorporate the same. Further, the references citedherein, such as Maniatis, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, p. 412 (1982), provide such techniques.

A gene (polynucleotide) can be used which encodes a corresponding orvariant polymerase having at least 80% homology to amino acid residues13-555 of SEQ ID NO: 26. These genes (polynucleotides) can encodevarious mutations. For example, a mutation of one or more amino acids inamino acids 461-490 of SEQ ID NO: 26 (738 to 767 of the Taq polymerasewild-type sequence SEQ ID NO: 100). Further examples of mutationsinclude mutations at positions M470, F472, M484, and W550, A331, andS335. In a preferred embodiment, the mutation may be at least one ofH203, F205, T232, E253, Q257, D274, L275, I276, V309, I322, A331, L332,D333, Y334, S335, I361, R374, A384, T387, Y419, P493, M498, G499, M502,L503, V506, R518, A523, A526, P539, E543, and W550.

In a preferred embodiment, these mutations are A331T, S335N, M470K,M470R, F472Y, M484V, M484T, and W550R. In a particularly preferredembodiment, the polynucleotides of the present invention encodepolypeptides having one or more of the aforementioned mutations andshare at least 85% identity, at least 90% identity, at least 95%identity, or at least 97.5% identity to the polypeptide comprising aminoacid residues 13-555 of SEQ ID NO: 26. Moreover, polynucleotides of thepresent invention encode polypeptides that have DNA polymerase activityand/or 5′-3′ exonuclease activity. More particularly, thepolynucleotides of the present invention encode polypeptides that arecapable of catalyzing the reverse transcription of RNA.

In the present invention, the polynucleotide may encode a polypeptidecontain at least one mutation at a position selected from the groupconsisting of A331, L332, D333, Y334, and S335.

The polynucleotide may encode a polypeptide of the present inventionwhich has amino acid sequence of residues 13-555 of a sequence selectedfrom the group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, and SEQ ID NO: 38.

Within the context of the present application, the preferredpolynucleotides possess a polynucleotide sequence corresponding tonucleotides 39-1667 of a sequence selected from the group consisting ofSEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37.

In another embodiment of the present invention, are thermostablepolypeptides having at least 80% homology, preferably at least 90%, morepreferably at least 95%, most preferably at least 97.5%, to residues13-555 of SEQ ID NO: 26, wherein said polypeptide has at least onemutation selected from the group consisting of H203R (position 480 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), F205L (position482 of the Taq polymerase wild-type sequence SEQ ID NO: 100), T232S(position 509 of the Taq polymerase wild-type sequence SEQ ID NO: 100),E253G (position 530 of the Taq polymerase wild-type sequence SEQ ID NO:100), Q257R (position 534 of the Taq polymerase wild-type sequence SEQID NO: 100), D274G (position 551 of the Taq polymerase wild-typesequence SEQ ID NO: 100), L275H (position 552 of the Taq polymerasewild-type sequence SEQ ID NO: 100), L275P (position 552 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), I276F (position 553 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), V309I (position586 of the Taq polymerase wild-type sequence SEQ ID NO: 100), I322N(position 599 of the Taq polymerase wild-type sequence SEQ ID NO: 100),A331V (position 608 of the Taq polymerase wild-type sequence SEQ ID NO:100), S335N (position 612 of the Taq polymerase wild-type sequence SEQID NO: 100), I361F (position 638 of the Taq polymerase wild-typesequence SEQ ID NO: 100), R374Q (position 651 of the Taq polymerasewild-type sequence SEQ ID NO: 100), A384T (position 661 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), T387A (position 664 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), Y419C (position696 of the Taq polymerase wild-type sequence SEQ ID NO: 100), Y419N(position 696 of the Taq polymerase wild-type sequence SEQ ID NO: 100),E465K (position 742 of the Taq polymerase wild-type sequence SEQ ID NO:100), M470K (position 747 of the Taq polymerase wild-type sequence SEQID NO: 100), M470R (position 747 of the Taq polymerase wild-typesequence SEQ ID NO: 100), F472Y (position 749 of the Taq polymerasewild-type sequence SEQ ID NO: 100), F472S (position 749 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), A487T (position 764 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), K490E (position767 of the Taq polymerase wild-type sequence SEQ ID NO: 100), P493T(position 770 of the Taq polymerase wild-type sequence SEQ ID NO: 100),M498T (position 775 of the Taq polymerase wild-type sequence SEQ ID NO:100), G499E (position 776 of the Taq polymerase wild-type sequence SEQID NO: 100), M502K (position 779 of the Taq polymerase wild-typesequence SEQ ID NO: 100), L503P (position 780 of the Taq polymerasewild-type sequence SEQ ID NO: 100), V506I (position 783 of the Taqpolymerase wild-type sequence SEQ ID NO: 100), A523V (position 800 ofthe Taq polymerase wild-type sequence SEQ ID NO: 100), A526V (position803 of the Taq polymerase wild-type sequence SEQ ID NO: 100), P539S(position 816 of the Taq polymerase wild-type sequence SEQ ID NO: 100),E543K (position 820 of the Taq polymerase wild-type sequence SEQ ID NO:100), and W550R (position 827 of the Taq polymerase wild-type sequenceSEQ ID NO: 100), and wherein said polypeptide has improved DNApolymerase activity and retains 5′-3′ exonuclease activity. In an objectof the present invention, the 3′-5′ exonuclease activity of the mutantpolypeptide is inactive.

Moreover, the present invention provides for polynucleotides that encodefor the aforementioned thermostable polypeptides within this embodiment.

In the present invention, the polynucleotide may encode a polypeptidehaving a sequence of residues 1-543 from a sequence selected from thegroup consisting of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ IDNO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ II) NO: 77, SEQID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87,SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:97, and SEQ ID NO: 99.

Within the context of the present application, the preferredpolynucleotides possess a polynucleotide sequence corresponding tonucleotides 1-1629 of a sequence selected from the group consisting ofSEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ IDNO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ IDNO:98.

In another embodiment of the present invention, the mutant DNApolymerase has a sequence corresponding to residues 1-543 of one of thefollowing sequences: SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ IDNO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87,SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:97, and SEQ ID NO: 99.

Within the scope of the present invention are also polynucleotides thatare homologous to the aforementioned sequences. In the context of thepresent application, a polynucleotide sequence is “homologous” with thesequence according to the invention if at least 80%, preferably at least90%, more preferably 95%, and most preferably 97.5% of its basecomposition and base sequence corresponds to the sequence according tothe invention. It is to be understood that, as evinced by the Examplesof the present invention and the phage-display method highlightedherein, screening of theoretical mutations within the scope of thepresent invention would require nothing more than a technician's levelof skill in the art. More specifically, as is routine in the art, withthe identification of a candidate sequence the artisan would assay andscreen one or all possible permutations of the said sequence to identifymutants possessing the same or better DNA polymerase activity, reversetranscriptase activity, and/or 5′-3′ exonuclease activity.

The expression “homologous amino acids” denotes those that havecorresponding properties, particularly withh regard to their charge,hydrophobic character, steric properties, etc.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may be determined conventionally by using knownsoftware or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wis. Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similaritybetween two sequences. Gap perfoms global alignments: all of onesequence with all of another similar sequence using the method ofNeedleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using asequence alignment program such as BestFit, to determine the degree ofsequerice homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores. Similarly, when using a programsuch as BestFit to determine sequence identity, siinilarity or homologybetween two different amino acid sequences, the default settings may beused, or an appropriate scoring matrix, such as blosum45 or blosum80,may be selected to optimize identity, similarity or homology scores.

The terms “isolated” or “purified” means separated from its naturalenvironment.

The term “polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, and can denote an unmodified RNA or DNA or amodified RNA or DNA.

The term “polypeptides” is to be understood to mean peptides or proteinsthat contain two or more amino acids that are bound via peptide bonds. A“polypeptide” as used herein is understood to mean a sequence of severalamino acid residues linked by peptide bonds. Such amino acids are knownin the art and encompass the unmodified and modified amino acids. Inaddition, one or more modifications known in the art such asglycosylation, phosphorylation, etc may modify the polypeptide.

The term “homologous” as used herein is understood to mean two or moreproteins from the same species or from a different species. Within themeaning of this term, said two or more polypeptides share at least 80%identity to residues 13-555 of the polypeptide of SEQ ID NO: 26 and canhave the mutations discussed herein. In a particularly preferredembodiment, the polypeptides of the present invention have one or moreof the aforementioned mutations and share at least 85% identity, atleast 90% identity, at least 95% identity, or at least 97.5% identity toresidues 13-555 of the polypeptide of SEQ ID NO: 26. Moreover, thepolypeptides of the present invention have DNA polymerase activityand/or 5′-3′ exonuclease activity. More particularly, the polypeptidesof the present invention are capable of catalyzing the reversetranscription of mRNA.

In the present invention, the polypeptide may contain one or moremutations, such as A331, L332, D333, Y334, and S335.

The isolated polypeptide of the present invention has an amino acidsequence corresponding to residues 13-555 of a sequence selected fromthe group consisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,and SEQ ID NO: 38.

Further, the mutant DNA polymerase may have a sequence corresponding toresidues 1-543 of one of the following sequences: SEQ ID NO: 63, SEQ IDNO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83,SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO:93, SEQ ID NO: 95, SEQ ID NO: 97, and SEQ ID NO: 99.

In an embodiment of the present invention are mutations concerningalanine in position 331 (A331), and serine in position 335 (S335) thatmay have particular importance derived from the fact that they aresurrounding the aspartic acid D in position 333 which is responsible forthe chelation of Mn²⁺ or Mg²⁺. Thus, in one embodiment of the presentinvention, mutations of one or more amino acids 10 amino acids upstreamand/or 10 amino acids downstream of this site are provided.

The expression “homologous amiino acids” denotes those that havecorresponding properties, particularly with regard to their charge,hydrophobic character, steric properties, etc.

Moreover, one skilled in the art is also aware of conservative aminoacid replacements such as the replacement of glycine by alanine or ofaspartic acid by glutamic acid in proteins as “sense mutations” which donot result in any fundamental change in the activity of the protein,i.e. which are functionally neutral. It is also known that changes atthe N- and/or C-terminus of a protein do not substantially impair thefunction thereof, and may even stabilize sai d function. As such, theseconservative amino acid replacements are also envisaged as being withinthe scope of the present invention.

The present invention also relates to DNA sequences that hybridize withthe DNA sequence that encodes a corresponding or variant polymerasehaving at least 80% homology, preferably at least 90%, more preferablyat least 95%, most preferably at least 97.5%, to residues 13-555 of SEQID NO: 26, the polypeptides having the mutations described herein. Thepresent invention also relates to DNA sequences that are produced bypolymerase chain reaction (PCR) using oligonucleotide primers thatresult from the DNA sequence that encodes a corresponding or variantpolymerase having at least 80% homology, preferably at least 90%, morepreferably at least 95%, most preferably at least 97.5%, to residues13-555 of SEQ ID NO: 26, wherein the polypeptide has at least onemutation as described herein, or fragments thereof. Oligonucleotides ofthis type typically have a length of at least 15 nucleotides.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). As used herein,stringent hybridization conditions are those conditions which allowhybridization between polynucleotides that are 80%, 85%, 90%, 95%, or97.5% homologous as determined using conventional homology programs, anexample of which is UWGCG sequence analysis program available from theUniversity of Wisconsin. (Devereaux et al., 1984). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some maismatching insequences so that lower degrees of similarity are detected heterologousprobing).

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperatLre is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemrplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with approximately 90% identity are sought, the Tm can bedecreased 10° C. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lonwer than the thermal melting point (Tm); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (Tm); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14,15, or 20° C. lower than the thermal melting point (Tm). Using theequation, hybridization and wash compositions, and desired Tm, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a Tm of less than 45° C.(aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inCurrent Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (2000).

Thus, with the foregoing information, the skilled artisan can identifyand isolated polynucleotides, which are substantially similar to thepresent polynucleotides. In isolating such a polynucleotide, thepolynucleotide can be used as the present polynucleotide in, forexample, to express a polypeptide having DNA polymerase activity and5′-3′ exonuclease activity.

One embodiment of the present invention is methods of screening forpolynucleotides, which have substantial homology to the polynucleotidesof the present invention, preferably those polynucleotides encoding apolypeptide having DNA polymerase activity and/or 5′-3′ exonucleaseactivity.

The polynucleotide sequences of the present invention can be carried onone or more suitable plasmid vectors, as known in the art for bacteriaor the like.

Host cells useful in the present invention include any cell having thecapacity to be infected or transfected by phages or vectors comprisingthe polynucleotide sequences encoding the enzymes described herein and,preferably also express the thermostable enzymes as described herein.Suitable host cells for expression include prokaryotes, yeast, archae,and other eukaryotic cells. Appropriate cloning and expression vectorsfor use with bacterial, fungal, yeast, and mammalian cellular hosts arewell known in the art, e.g., Pouwels et al. (1985). The vector may be aplasmid vector, a single or double-stranded phage vector, or a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsulated virus by well-known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems could also be employed to produce the enzymes using RNAs derivedfrom the present DNA constructs.

Prokaryotes useful as host cells in the present invention include gramnegative or gram-positive organisms such as E. coli or Bacilli. In aprokaryotic host cell, a polypeptide may include a N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-terminal Met may be cleaved from theexpressed recombinant polypeptide. Promoter sequences commonly used forrecombinant prokaryotic host cell expression vectors include β-lactamaseand the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means fox identifyingtransformed cells. To construct an expression vector using pBR322, Onappropriate promoter and a DNA sequence are inserted into the pBR322vector.

Other commercially available vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec,Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., 1978; and Goeddel et al., 1979), tryptophan (trp)promoter system (Goeddel et al., 1980), and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412 (1982)).

Yeasts useful as host cells in the present invention include those fromthe genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., 1980)or other glycolytic enzymes (Holland et al., 1978) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvateedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer etal., Gene, 107:285-195 (1991). Other suitable promoters and vectors foryeast and yeast transformation protocols are well known in the art.

Those of skill in the art are familiar with yeast transformationprotocols that may be employed in the present invention. One suchprotocol is described by Hinnen et al., (1978). The Hinnen protocolselects for Trp⁺ transformants in a selective medium, wherein theselective medium consists of 0.67% yeast nitrogen base, 0.5% casaminoacids, 2% glucose, 10 μg/ml adenine, and 20 μg/ml uracil.

Mammalian or insect host cell culture systems well known in the artcould also be employed to express recombinant polypeptides, e.g.,Baculovirus systems for production of heterologous proteins in insectcells (Luckow and Summers, (1988)) or Chinese hamster ovary (CHO) cellsfor mammalian expression may be used. Transcriptional and translationalcontrol sequences for mammalian host cell expression vectors may beexcised from viral genomes. Commonly used promoter sequences andenhancer sequences are derived from Polyoma virus, Adenovirus 2, SimianVirus 40 (SV40), and human cytomegalovirus. DNA sequences derived fromthe SV40 viral genome may be used to provide other genetic elements forexpression of a structural gene sequence in a mammalian host cell, e.g.,SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites. Viral early and late promoters are particularlyuseful because both are easily obtained from a viral genome as afragment which may also contain a viral origin of replication. Exemplaryexpression vectors for use in mammalian host cells are well known in theart.

The enzymes of the present invention may, when beneficial, be expressedas a fusion protein that has the enzyme attached to a fusion segment.The fusion segment often aids in protein purification, e.g., bypermitting the fusion protein to be isolated and purified by affinitychromatography. Fusion proteins can be produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of the enzyme.

In one embodiment, it may be advantageous for propagating thepolynucleotide to carry it in a bacterial or fungal strain with theappropriate vector suitable for the cell type. Common methods ofpropagating polyrn-ucleotides and producing proteins in these cell typesare known in the art and are described, for example, in Maniatis et al.(1982) and Sambrook et al. (1989).

In one embodiment of the present invention are monoclonal phages:

-   -   1. SJL q deposited under Budapest treaty as CNCM I-3168 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France), on Feb. 27, 2004.    -   2. SJL d deposited under Budapest txeaty as CNCM I-3169 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   3. SJL I deposited under Budapest treaty as CNCM I-3170 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   4. SJL s deposited under Budapest treaty as CNCM I-3171 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   5. SJL b deposited under Budapest txeaty as CNCM I-3172 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   6. SJL n deposited under Budapest treaty as CNCM I-3173 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   7. SJL g deposited under Budapest treaty as CNCM I-3174 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   8. SJL m deposited under Budapest treaty as CNCM I-3175 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   9. SJL a deposited under Budapest treaty as CNCM I-3176 in the        Collection Nationale de Cultures de Microorganismes (CNCM,        Institut Pasteur 25 rue du docteur Roux 75724 Paris cedex 15        France) on Feb. 27, 2004.    -   10. SVG VIII-176 deposited as CNCM I-3158 in the Collection        Nationale de Cultures de Microorganismes (CNCM) on Feb. 10,        2004.

In an embodiment of the present invention is a kit for amplifying DNAcontaining:

-   -   an isolated thermostable polypeptide, wherein said polypeptide        has at least 80% homology to residues 13-555 of SEQ ID NO: 26,        wherein said polypeptide has at least one mutation at a position        selected from the group consisting of M470, F472, M484, R518,        and W550, more preferably selected from the group consisting of        M470K, M470R, F472Y, M484V, M484T, R518G, and W550R, and wherein        said polypeptide has DNA polymerase activity and 5′-3′        exonuclease activity;    -   a concentrated buffer solution, whereiii when said concentrated        buffer is admixed with the isolated polypeptide the overall        buffer concentration is 1X;    -   one or more divalent metal ions; and    -   deoxyribonucleotides.

In this embodiment, the preferred divalent metal ion is Mg²⁺. In another embodiment, the metal ion may also be Mn²⁺. In this connection,the concentration of the divalent metal ion ranges from 0.1 to 5 mM,preferably from 1 to 3 mM, more preferably from 2 to 2.5 mM. However, ifthe reaction is performed in a phosphate buffer, a buffer containingEDTA, or a buffer containing any other magnesium chelator, theconcentration of magnesium may be increased to up to 100 mM.

For the kit of the present invention the isolated thermostablepolypeptide may be in a form selected from the group consisting of alyophilized form, a solution form in a suitable buffer or carrier, and afrozen form in a suitable buffer or carrier.

The kit of the present invention may also include a 5′ to 3′ exonucleaseand/or a 3′ to 5′ exonuclease. A preferred 5′ to 3′ exonuclease has asequence as in SEQ ID NO: 50 (the DNA is in SEQ ID NO: 60) and the 3′ to5′ exonuclease as in SEQ ID NO: 51 (the DNA is in SEQ ID NO: 61).

With respect to the suitable buffer or carrier, the following componentsmay be used:

-   Tris-HCl, KCl, Triton-X100, dimethylsulfoxide, tetramethyl ammonium    chloride, etc.

In the present invention, the concentrated buffer solution correspondsto a stock solution that has a concentration ranging from 1.5X to 10X,where the concentration is measured in relation to the final reactionconcentration (1X). To this end, the buffer solution (1X) contains thefollowing components: 10 mM Tris-HCl, pH at 25° C. of 9, 50 mM KCl, 0.1%Triton-X100.

For the kit according to the present invention, the stock concentrationof the deoxyribonucleotides ranges from 50 μM to 200 mM, preferably from75 μM to 150 mM, more preferably 100 μM to 100 mM, for each dNTP.Moreover, the concentration of each dNTP in the PCR reaction accordingto the present invention should range from 10 μM to 500 μM, preferablyfrom 25 μM to 400 μM, more preferably 50 μM to 300 μM. As used in thepresent invention, tILe term “deoxyribonucleotides” includes: dATP,dCTP, dGTP, and dTTP. It is to be understood that within the scope ofthe present invention, the kit may include in place of or in addition tothe aforementioned components, RNA precursors, minor (“rare”) bases,and/or labelled bases.

In another embodiment of the present invention is a method of amplifyingDNA from a culture and/or purified stock solution of DNA and/or mRNA byutilizing a thermostable polypeptide according to the present invention.To this end, protocols for conducting PCR and RT-PCR would be readilyappreciated by the skilled artisan. However, for sake of completeness,the artisam is directed to the following exemplary references forprotocols for conducting PCR and RT-PCR (see, for example, Rougeon, F,et al.; 1975; Rougeon, F, et al., 1976; Grablco, V. I., et al., 1996;and Perler, F., et al., 1996).

With reference to reverse transcribing RNA, a preferred method includes:

-   -   a) providing a reverse transcription reaction mixture comprising        said RNA, a primer, a divalent cation, and an isolated        thermnostable polypeptide comprising an amino acid sequence        having at least 80% homology to residues 13-555 of SEQ ID NO:        26, wherein said polypeptide has at least one mutation at a        position selected from the group consisting of H203, F205, T232,        E253, Q257, D274, L275, I276, V309, I322, A331, L332, D333,        Y334, S335, I361, R374, A384, T387, Y419, M470, F472, p484,        P493, M498, G499, M502, L503, V506, R518, A523, A526, P539,        E543, and W550, more preferably selected from the group        consisting of: M470K, M470R, F472Y, M484V, M484T, R518G, and        W550R; or H203R, F205L, T232S, E253G, Q257R, D274G, L275H,        L275P, I276F, V309I, I322N, A331V, S335N, I361F, R374Q, A384T,        T387A, Y419C, Y419N, E465K, M470K, M470R, F472Y, F472S, A487T,        K490E, P493T, M498T, G499E, M502K, L503P, V506I, R518G, A523V,        A526V, P539S, E543K, and W550R, and wherein said polypeptide has        DNA polymerase activity and 5′-3′ exonuclease activity in a        suitable buffer; and    -   b) treating said reaction mixture at a temperature and under        conditions suitable for said isolated polypeptide to initiate        synthesis of an extension product of said primer to provide a        cDNA molecule complementary to said RNA.

It is to be understood that the skilled artisan would appreciate thatthe thermal cycling should be optimized to account for variations in theenzyme selected, the template to be reverse transcribed, the primers tobe used to facilitate amplification (i.e., with respect to the meltingand annealing temperatures), and the relative concentrations to be usedfor each of the reaction components. Such optimization is well withinthe purview of the skilled artisan; however, exemplary protocols mayinclude the following:

TABLE 2 PCR protocols # of repeated a b c d e Cycles PCR 1 94° C., 3′94° C., 1′ 66° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 30 PCR 2 94° C., 3′94° C., 1′ 62° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 30 PCR 3 94° C., 3′94° C., 30″ 59° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 30 PCR 4 94° C.,3′ 94° C., 30″ 68° C., 1.5′ 68° C., 6′ b − c = 35 PCR 5 94° C., 1′ 94°C., 30″ 70° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 25 PCR 6 94° C., 3′94° C., 30″ 59° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 35 PCR 7 94° C.,3′ 94° C., 1′ 58° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 35

Moreover, it is to be understood that contemplated in the presentinvention is that with the polypeptide of the present invention theskilled artisan would appreciate that the buffer components and bufferconcentrations should also be optimized. To this end, in a preferredembodiment, the kit of the present invention may be utilized.

As used above, the phrases “selected from the group consisting of,”“chosen from,” and the like include mixtures of the specified materials.

The term ‘selection’ relates to the parallel processing of a variants'catalytic activity (cf. affinity chromatography for the reaction productcrosslinked to active phage-polymerase mutants allows the ‘simultaneous’treatment of the population of phage-polymerase mutants). Selection canbe achieved for a population of more than 10⁷ mutants as describedherein, for more than 10¹⁰ variants and possibly for up to 10¹⁴variants. A straightforward screening of several tens of variants of theselected population enriched into active enzymes is sufficient toisolate catalysts of interest.

The term ‘screening’ relates to the serial processing of a variants'catalytic activity (cf. serial assays ran by a robot one well after thenext one). High throughput-screening is typically done for 10⁴ mutants,and generally less than 10⁷ mutants.

The advantage of selection over high throughput screening is that a muchlarger population of mutant proteins can be analyzed for a desiredcatalytic activity; provided an appropriate selection strategy isavailable (the one described herein is one such example).

In one embodiment of a method of obtaining a thermostable variant enzymeis provided. This method comprises the following:

-   -   a) selection of enzymes expressed at the surface of phage        particles and identifying at least a thermostable variant        conserving its active; catalytic domain at regulated temperature        according to the method of identifying thermostable mutant        polypeptides having a catalytic activity as described herein,    -   b) isolating and sequencing a DNA encoding said identified        thermostable variant;    -   c) preparing a vector comprising the DNA of step (b);    -   d) transfecting or infecting cells with the vector obtained at        step c);    -   e) expressing the thermostable variant enzyme from the cells and        optionally,    -   f) recovering, isolating and purifying said thermostable variant        enzyme expressed at step (e).

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Materials and Methods

Buffers

Buffer A (1×):

-   -   50 mM Tris-HCl at pH 8.3 at 25° C., 50 mM KCl, 10 mM MgCl₂, 0.5        mM spermidine, 10 mM dithiothreitol

Buffer B (1×):

20 mM Tris-HCl at pH 8.8 at 25° C., 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mMMgSO₄, 0.1% Triton X-100, 0.1 g/l BSA

Buffer C (1×):

-   -   10 mM Tris-HCl at pH 9.0 at 25° C., 50 mM KCl, 0.1% Triton X-100

Synthesis of Substrates for Selection

Deoxyoligonucleotides were prepared by solid phase synthesis on a DNAsynthesizer (Expedite™, Millipore). The 5′-maleimidyl derivatized primerTAA CAC GAC AAA GCG CAA GAT GTG GCG T (SEQ ID NO: 13) was synthesized asdescribed previously (Jestin et al., 1999) purified on a C18 reversephase HPLC column, and characterized by electrospray mass spectroscopy8998.4/8999.9 (measured/calculated).5-[-N-[N-(N-biotinyl-ε-aminocaproyl)-γ-aminobutyryl]-3-aminoallyl]-2′deoxy-uridine-5′-triphosphate(biotin-dUTP) was purchased from Sigma and the other deoxynucleotidetriphosphates dATP, dCTP and dGTP were obtained from Roche-Boehringer.

Library Construction

Three phagemids libraries were mixed for phage preparation. The firsttvo libraries (I: FseI/NotI and II: PstI/NheI) derive from mutagenic PCRamplification of the wild-type Taq gene in the presence of manganese [I:reference (Fromant, Blanquet, Plateau, 1995) with MnCl₂: 0.5 mM; II:reference (Cadwell, Joyce, PCR methods and amplifications, MutagenicPCR, 3, S136-S140) with four distinct MnCl₂ concentrations (0.5, 0.35,0.25 and 0.125 mM)] using following primers (I) SEQ ID NO: 1 and SEQ IDNO: 2, PCR 1, or (II) SEQ ID NO: 3 and SEQ ID NO: 4, PCR 2 (for primers:see Table 1, and for cycle settings: see Table 2).

The third phagemids library (III) was constructed by oligonucleotideassembly using the wild-type Taq gene. First, four PCR fragments wereprepared using laq polymerase (PCR 3, see Table 2), the wild-typeStoffel fragment gene as template and the following primer pairs (5-6),(7-8), (9-10) and (11-2) in buffer C 1X (for primers: see Table 1).

After purification with the QIAquick PCR Purification kit (QIAGEN), thefour PCR fragments were assembled in a second PCR round using the kitGC-Advantage obtained from Clontech under PCR 4 (see Table 2), usingbuffer D 1X. The crude PCR product was then amplified by PCR using PCR 5protocol, the GC-Advantage kit, and the primers 1 and 2 in buffer D 1X.Subsequently, the product was purified using the QIAquick Gel extractiongel (QIAGEN).

Buffer D 1X

-   -   40 mM Tricine-KOH (pH 9.2)    -   15 mM KOAc    -   3.5 mM Mg(OAc)₂    -   5% DMSO    -   3.75 μg/ml BSA    -   0.005% Nonidet P-40    -   0.005% Tween-20

After subdloning into pHENI vectors using restriction sites FseI/NotI orPstI/NheI, 1.1×10⁷ distinct clones were obtained by electroporation inE. coli strain TG1.

TABLE 1 Oligonuleotides and primers SEQ ID NO: Oligonucleotide sequences1 TAACAATAGGCCGGCCACCCCTTC 2 GAGTTTTTGTTCTGCGGC 3TTTAATCATCTGCAGTACCGGGAGCTC 4 TTCATTCTTGCTAGCTCCTGGGAGAGGC 5 CCG GCC ACCCCT TC(C AR/A VY)C TCA AC(C AR/A VY)CGG GAC CAG CTG GAA AG 6 GGA TGA GGTCCG GCA A(YT G/RB T) (YT G/RB T)AA T(YT G/RB T)GG TGC T CT TCA GCT T(YTG/RB T)GA GCT CCC GGT ACT GCA GG 7 CAA CCA GAC GGC CAC G(CA R/AV Y)ACGGG CAG GCT A(CA R/AV Y)AG CTC C(CA R/AV Y)CC CAA CCT CCA GAA CAT CC 8CCG CCT CCC GCA C(YT G/RB T)CT TCA C(YT G/RB T)GG CCT CTA GGT CTG GCA C9 CCT GCA GTA CCG GGA GCT C(CA R/AV Y)AA GCT GAA GAG CAC C (CA R/AV Y)ATT(CA R/AV Y)(CA R/AV Y)TT GCC GGA CCT CAT CC 10 GGA TGT TCT GGA GGT TGGG(YTG/RBT)GG AGC T(YTG/ RBT)TA GCC TGC CCG T(YTG/RBT)CG TGG CCG TCT GGTTG 11 GTG CCA GAC CTA GAG GCC (CAR/AVY) GTG AAG (CAR/ AVY) GTG CGG G AGGCG G 12 AAA UAC AAC AAU AAA ACG CCA CAU CUU GCG 13 TAA CAC GAC AAA GCGCAA GAT GTG GCG T 14 AAA TAC AAC AAT AAA ACG CCA CAT CTT GCG 15TTCATTCTTGCTAGCTCCTGGGAGAGGC 16 GAG AAG ATC CTG CAG TAC CGG GAG C 17GACCAAC ATCAAGACTGCC 18 TTGGCCAGGAACTTGTCC

TABLE 2 PCR cycles # of repeated a b c d e Cycles PCR 1 94° C., 3′ 94°C., 1′ 66° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 30 PCR 2 94° C., 3′ 94°C., 1′ 62° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 30 PCR 3 94° C., 3′ 94°C., 30″ 59° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 30 PCR 4 94° C., 3′94° C., 30″ 68° C., 1.5′ 68° C., 6′ b − c = 35 PCR 5 94° C., 1′ 94° C.,30″ 70° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 25 PCR 6 94° C., 3′ 94°C., 30″ 59° C., 30″ 72° C., 1′ 72° C., 15′ b − d = 35 PCR 7 94° C., 3′94° C., 1′ 58° C., 1′ 72° C., 2′ 72° C., 15′ b − d = 35

Phage Preparation and Selection

For phage preparation, E. coli TG1 transformed by the phagemid libraryand grown to an optical density of 0.3 at 600 nm were infected by atwenty-fold excess of helper phage. Phage particles were produced at 30°C. for 19 hours in a 2×TY medium containing 100 mg/l ampicillin, 25 mg/lkanamycin. After removal of bacteria by two centrifugation (4000 rpm, 4°C.), phage particles in the supernatant were purified by twoprecipitations in 4% polyethyleneglycol in 0.5 M NaCl, resuspended in 1ml of PBS (25 mM Na₂HPO₄, 100 mM NaCl, pH 7.4), and dialyzed four timesagainst PBS over a period of 24 hours. The pH of the final solution wasraised to pH 8.

The protocol for selection was as described previously (Jestin et al.,1999), except that 10¹⁰ infectious phages particles were used afterheating at 65° C. for 5 minutes and that DNA polymerization was done at65° C. for 2 minutes.

Substrate cross-linking on phage was done by incubating the phageparticles with 10 μM maleimidyl-derivatized primer, 50 μM RNA templateof SEQ ID NO: 12 in the presence of 10 mM magnesium chloride at 37° C.for 2 hours and polymerization during 2 minutes at 65° C. after additionof 3 μM biotin-dUTP and 1 μM dVTP.

The reactions were blocked by addition of one volume of 0.25 M ethylenediamine tetra-acetate. The phage mixture was added to 300 μlofstreptavidin-coated superparamagnetic beads (Dynabeads M-280, Dynal).After 30 minutes at room temperature, the beads were washed seven timesand resuspended in 200 μl PBS.

The phage-bead mixture was incubated for 10 min at 37° C. after additionof one-tenth, in volume, of trypsin (0.1 g/l). 1.8 mL of E. coli TG1 wasthen added for infection during 25 min at 37° C. Bacteria were plated on530 cm² Petri dishes (Corning) with a 2×TY medium containing ampicillin(0.1 g/1). After 12 hours at 30° C., bacteria were scraped from theplates and about 2×10⁹ cells were used for preparation of the phageparticles.

Variant Polymerases

All variant Stoffel fragment sequences appearing in the followingexamples correspond to residues 13-555 of SEQ ID NO: 26, which in turncorrespond to residues 290-832 of SEQ ID NO: 100, which contain one ormore mutations. A summary of the mutations, the sequence designator(e.g., “s,” “a,” “m,” etc.), and the sequence identifier (i.e., SEQ IDNO) appear in the Table at the end of the Examples. Each of the variantsin the following examples contain a R795G mutation (i.e., residuecorresponding to 518 in the sequence of SEQ ID NO: 26); however, thismutation need not be present within the context of the presentinvention. In other words, the present invention embraces variantscorresponding to the above in which R795 is conserved. It should benoted that the only mutation appearing in variant e is the R795Gmutation, otherwise this sequence corresponds to the wild-type Stoffelfragment (residues 13-555 of SEQ ID NO: 26).

RT-polvmerization and polymerization activity assay usingphage-polvmerase

In the following examples, the activity of the different mutantphage-polymerases was assayed by incorporation of radiolabeled alpha ³²PdTTP.

Example 1 Polyclonal Phage-polymerases (FIG. 1):

In this example, the reverse transcriptase activity of phage-polymeraseswas assessed in the presence of Mg²⁺ or Mn²⁺ ions as obtained afterdifferent rounds of selection in the presence of Mg²⁺ ions. In theseexperiments, two reverse transcription (RT) mixes were used. The finalconcentration of each component in a reaction was: 10 μM RNA (SEQ ID NO:12); 5 μM DNA (SEQ ID NO: 13); 0.25 mM dNTP; 3 mM MgCl₂ or 2.5 mM MnCl₂.

Each 1.9 μl aliquot of the reaction mix was further added to 15 μl ofphage-polymerases (about 10⁸ particles) after a given selection roundheated for 5 min at 65° C. The solutions were then incubated at 37° C.for 15 min. The reactions were stopped by adding 15 μl of EDTA/formamidecontaining denaturation solution, heating for 3 min. at 94° C., andplaced on ice. The incorporation of alpha ³²P-dTTP was determined on 20%polyacrylamide gel; 15 μl of the final reaction volume were loaded.

The lane designations in FIG. 1 are as follows:

MnCl2 MgCl₂ a: phage-polymerases of round 6 h: phage-polymerases ofround 6 b: phage-polymerases of round 5 i: phage-polymerases of round 5c: phage-polymerases of round 4 j: phage-polymerases of round 4 d:phage-polymerases of round 3 k: phage-polymerases of round 3 e:phage-polymerases of round 2 1: phage-polymerases of round 2 f:phage-polymerases of round 1 m: phage-polymerases of round 1 g:phage-polymerases of initial n: phage-polymerases of initial populationpopulation

This experiment demonstrated that:

-   -   A RT-activity is present using phage-polymerase obtained after        round 5 (i) or 6 (h) of selection in presence of Mg²⁺.    -   A high RT-activity was detected at the round 3 (d) in the        presence of Mn²⁺ and for further rounds.

Example 2 Polyclonal Phage-polymerases (FIG. 2)

In this example, the reverse transcriptase activity of phage-polymeraseswas assessed as obtained after different rounds of selection in thepresence of Mg²⁺ ions. In these experiments, a reverse transcription(RT) mix was used. The final concentration of each component in areaction was: 10 μM RNA (SEQ ID NO: 12); 5 μM DNA (SEQ ID NO: 13); 0.25mM dNTP; 3 mM MgCl₂.

Each 1.2 μl aliquot of the reaction mix was further mixed with 15 μl ofphage-polymerase polymerases (about 10⁸ particles) after one round ofselection round, either not preheated or heated 5 min at 65° C. beforereaction of polymerization. The solutions were then incubated at 37° C.for 15 min. The reactions were stopped by adding 15 μl of thedenaturation solution, heating for 3 min. at 94° C. and placing on ice.

The incorporation of alpha ³²P-dTTP was determined on 20% polyacrylamidegel; 15 μl of the final reaction volume were loaded. The positivecontrol was performed with addition of different concentration ofcommercial AMV reverse transcriptase (Promega).

The lane designations in FIG. 2 are as follows:

Phage-porymerase preheated at 65° C. for 5 min. Phage-polymerase notpreheated a: phage-polymerases of initial h: phage-polymerases ofinitial population population b: phage-polymerases of round 1 i:phage-polymerases of round 1 c: phage-polymerases of round 2 j:phage-polymerases of round 2 d: phage-polymerases of round 3 k:phage-polymerases of round 3 e: phage-polymerases of round 4 1:phage-polymerases of round 4 f: phage-polymerases of round 5 m:phage-polymerases of round 5 g: phage-polymerases of round 6 n:phage-polymerases of round 6 o: control AMV-RT, 1 U p: control AMV-RT,0.1 U q: control AMV-RT, 0.01 U r: control AMV-RT, 0.001 U

This experiment demonstrated that:

-   -   A RT-activity is present using phage-polymerase obtained after        round 5 or 6 of selection preheated for 5 min. at 65° C. (f        and g) or not (m and n) as in FIG. 1 in presence of Mg²⁺.    -   A high RT-activity was detected using 1 unit of AMV-RT (o) but        no activity was detected using decreasing concentration of        AMV-RT.

Example 3 Monoclonal Phage-polymerases (FIG. 3)

In this example, the reverse transcriptase activity of variousmonoclonal phage-polymerases obtained after round 6 in the presence ofMg²⁺ ions was assessed. In these experiments, a reverse transcription(RT) mix was prepared in which the final concentration of each componentin a reaction was: 10 μM RNA (SEQ ID NO: 12); 5 μM DNA (SEQ ID NO: 13);0.25 mM dNTP; 3 mM MgCl₂.

Each 1.45 μl aliquot of the reaction mix was further mixed with 15 μl ofphage-polymerase heated for 5 min at 65° C. The solutions were thenincubated at 37° C. for 20 min. The reactions were stopped by adding 15μl of denaturation solution, heating for 3 min. at 94° C., and placed onice.

The incorporation of alpha ³²P-dTTP was determined on a 20%polyacrylamide gel; 15 μl of the final reaction volume were loaded. Thepositive control was performed using the AMV-RT (Promega), lane C.

The different monoclonal phage-polymerases were obtained among thephage-polymerases of round 6. The phage-polymerases present variousDNA-polymerase RNA-dependant activities. The lane designations in FIG. 3are as follows: s=SEQ ID NO: 38; a=SEQ ID NO: 20; d=SEQ ID NO: 24; g=SEQID NO: 28; C=AMV-RT; i=SEQ ID NO: 30; m=SEQ ID NO: 32; n=SEQ ID NO: 34;b=SEQ ID NO: 22; and q=SEQ ID NO: NO: 36.

The clones a, b, and d possess a high RT-activity, which were furtherstudied as reported in FIG. 4. Randomly chosen clones from the selectedpopulations were assayed for monoclonal phage-polymerase reversetranscriptase activity and that further sequencing of the correspondingmutant genes revealed identical sequences (for example, 7 clonesreported in the figure were found to have the same sequence noted a).

Example 4 Monoclonal Phage-polymerases (FIG. 4)

In this example, the reverse transcriptase and the polymerase activitiesof monoclonal phage-polymerases obtained after the round 6 in thepresence of Mg²⁺ or Mn²⁺ ions was assessed. In these experiments, thefinal concentration of each component in a reaction was:

10 μM RNA (SEQ ID NO: 12); 5 μM DNA (SEQ ID NO: 13); 0.25 mM dNTP; 3 mMMgCl₂ or 2.5 mM MnCl₂; and

1 μM DNA (SEQ ID NO: 14); 1 μM DNA (SEQ ID NO: 13); 0.25 mM dNTP; 3 mMMgCl₂ or 2.5 mM MnCl₂;

2 μl aliquots of the reaction mix were further added to 15 μl of eachphage-polymerase pre-heated for 5 min at 65° C. The solutions were thenincubated at 37° C. for 15 min. The reactions were stopped by adding 15μl of denaturation solution, heating 3 min. at 94° C., and placed onice.

The incorporation of alpha ³²P-dTTP was determined on polyacrylamidegel; 15 μl of the final reaction volume were loaded. The positivecontrol was performed using the phage Stoffel fragment (e).

The lane designations in FIG. 4 are as follows: a=SEQ ID NO: 20; b=SEQID NO: 22; d=SEQ ID NO: 24; and e=SEQ ID NO: 26 (containing an R518Gmutation).

Three families of phage polymerase were characterized among thephage-polymerases of round 6.

-   -   The phage-polymerases a and b present a high DNA-polymerase        DNA-dependent activity, which is higher than that of Stoffel        phage-polymerase.    -   The phage-polymerases b and d present a high DNA-polymerase        RNA-dependent activity, which is higher than that of the Stoffel        phage-polymerase e (not detectable, see FIG. 4) or than the        phage-polymerase a, whatever the conditions in the presence of        magnesium or of manganese.    -   The phage-polymerase d shows a poor DNA-polymerase DNA-dependent        activity, which is lower than the activity of the Stoffel        phage-polymerase.

Construction and Overproducing Clones

Three phagemids corresponding to clones a, b and d on FIG. 4 wereisolated from individual colonies of E. coli strain TG1. The plasmid DNAwas prepared and purified using Wizard Plus miniprep kits. The phagemidswere cleaved with NcoI and NotI restriction endonucleases and thefragments ligated into an expression vector deriving from pET-28b(+)(Novagen) that had been cleaved with NcoI and NotI and dephosphorylatedwith alkaline phosphatase. This pET vector contains a sequence for thethrombin cleavage site between the NotI and XhoI restriction sites ofpET 28b(+) (GCGGCCGCACTGGTGCCGCGCGGCAGCCTCGAG; SEQ ID NO: 45).

Recombinant plasmids were transformed in E. coli strain BL21(DE3) pLysSand plated on 2YT media with kanamycin and chloramphenicol. Correctplasmid constructions were initially identified by restriction analysisof plasmid miniprep.

E. coli strain BL21(DE3) pLysS, used as a host for recombinant plasmidsto over produce the mutant polymerase, was grown in 2YT mediumsupplemented with 10 μg/ml kanamycin and 25 μg/ml chloramphenicol topropagate plasmids. At an optical density of 0.6 at 600 nm, 1 mM ofisopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induceproduction of enzyme for 5 hours.

Purification of Mutant Polymerases

Mutants were prepared from 500 ml batches of cells. 2YT media pluskanamycin and chloramphenicol was inoculated with bacteria (containing arecombinant plasmid) freshly picked on a plate and grown at 37 ° C. toan absorbance at 600 nm of approximately 0.5. Subsequently, IPTG wasadded to a final concentration of 1 mM and the cultures were allowed tofurther grow for 5 h.

Cells were harvested by centrifugation at 15000 g and 4° C. for 10 min.,resuspended in 30 ml of lysis buffer (50 mM Na₂HPO₄, 300 mM NaCl, 5 mMimidazole, pH=8), lysed 3 times for 45 sec by ultrasound. Cell debriswere removed by centrifugation at 10000 g and 4° C. for 15 min.

Mutant polymerases were recovered from this clarified lysate andpurified using Ni-NTA agarose (QIAGEN).

Example 5 Purified Mutant Polymerases a, b, and d Used in RT-polymeraseChain Reaction (FIG. 5):

The positive control was performed using the polymerase AMV-RT(Promega). These studies were performed using the three clonescorresponding on clones a, b and d in FIG. 4.

The reverse transcription was performed at 65° C. during 1 h using thefollowing conditions.

Control RT mix Component Amount RNA from rabbit globin (sigma), 20 μg/ml1 μl primer 17 (5 μM) 0.4 μl primer 18 (5 μM) 0.4 μl buffer A (**)AMV-RT5× 3 μl dNTP 2.5 mM 0.8 μl AMV-RT 10 U/μl 3 μl water 6.4 μl (**) Seebuffer A composition above

RT mix Component Amount RNA from rabbit globin (sigma), 20 (μg/ml 1 μlprimer 17 (5 μM) 0.4 μl primer 18 (5 μM) 0.4 μl MgCl₂ 25 mM 0.75 bufferC (***) 1.5 μl dNTP 2.5 mM 0.8 μl mutant polymerase a, b, d 3 μl or theStoffel fragment e water 7.15 μl (***) See buffer C composition above

The PCR was performed using PCR 7 (see table 2) and followingconditions.

PCR mix Component Amount Buffer B 10× 20 μl primer 17 (50 μM) 4 μlprimer 18 (50 μM) 4 μl dNTP 2 μl water 164.5 μl Taq DNA polymerase (5U/μl) 5 μl Pfu polymerase (3 U/μl) 0.5 μl

19 μl aliquot of the PCR mix was added to 1 μl of the RT reactionproduct.

A RT-PCR product of 372 bp was detectable using mutant polymerases b andd.

The lanes in the gel appearing in FIG. 5 include the three clonescorresponding to clones a, b and d on FIG. 4. In addition, the positivecontrol was performed using the Stoffel fragment polymerase e and thecommercial AMV-RT (Promega). The lanes in FIG. 5 are as follows:

-   -   lane 1: molecular weight marker: PhiX phage DNA digested by        HaeIII.    -   lane 2: control AMV-RT    -   lane 3: b=SEQ ID NO: 22    -   lane 4: a=SEQ ID NO: 20    -   lane 5: e=SEQ ID NO: 26 (containing an R518G mutation)    -   lane 6: d=SEQ ID NO: 24

Example 6 Optimization of the production and of the purification of thepolymerases

Variant polymerases were expressed in Escherichia coli strain BL21(DE3)pLysS using a pET vector. The inventors improved the yield and thereproducibility of production of these proteins by (a) a chemical lysisof the cells and (b) a pre-purification by heating at 80° C. for 10 or15 minutes (denaturation, elimination by precipitation andcentrifugation of the proteins thermically unstable).

The optimization of the two steps (a) and (b) allows a more effectivepurification of variants by chromatography.

After affinity chromatography by using the six histidine tag, inventorsobtained, starting from about 1.2 to 1.6 liters of culture, until about0.1 to 0.3 mg of protein of purity, estimated on polyacrylamide gel,comprised between 80% and 90% Further purification steps were done priorto kinetic studies (see Example 10).

Protocol of Production and Purification of Proteins Production ofVariants d, b, a and e.

Electroporation of 50 μl of electro-competent cells BL21(DE3) pLysS bythe expression pET plasmid containing mutated or wild-type genes (e,fragment of Stoffel of the Taq polymerase) coding for the proteinspolymerases to be produced.

Incubation 1 hour at 37° C. in 3 ml of 2YT medium under agitation.

Spreading out with beads of 100 and 200 μl per Petri dish containing 2YTagarose with kanamycin (10 μg/ml) and chloramphenicol (30 μg/ml).

Incubation at 37° C. for 16 hours.

The colonies are recovered and suspended in 400 ml of 2YT medium withkanamycin (10 μg/ml) and chloramphenicol (30 μg/ml).

Incubation at 37° C. of the culture until the OD at 600 nm is about 0.6.

Induction of the expression of the proteins by addition of IPTG (1 mMfinal concentration).

Incubation about 16 hours at 37° C. under agitation.

Centrifugation of the cultures at 8000 rpm, 10 min., 4° C.

Purification

The pellets were treated by 45 ml of BugBuster (BugBuster ProteinExtraction Reagent-NOVAGEN, Ref. 70584).

Incubation at room temperature under agitation 20-40 min.

Centrifugation at 10000 rpm, 10 min., 21° C.

Supernatants were recovered and treated 10 min. at 80° C. underagitation prior to coiling at 4° C.

Centrifugation at 10000 rpm, 10 min., 4° C.

Capture of the Proteins by Affinity Chromatography for the His Tag

The Talon Metal Affinity Resin (2 ml., Clontech) was washed twice withthe buffer BBW (50 mM sodium phosphates, 300 mM NaCl, pH=7) prior toincubation for 30 minutes at 4° C. with the supernatant. After threewashing steps with the BBW buffer (20 ml.), the proteins were elutedfrom the resin.

Elution

Add to each resin pellet to be eluted, 2 ml of 1X Elution Buffer:hnidazole Elution (pH7, 50 mM Sodium Phosphates, 300 mM NaCl, 150 mMimidazole).

FRACTION 1: Homogenize 10 min. with at cold temperature and centrifuge700 g, 3 min., 4° C. Recover the supernatant (2 ml). Add 2 ml 1X ElutionBuffer, Homogenize 5 min.

FRACTION 2: Centrifuge 700 g, 3min., 4° C. Recover supernatant (2ml ).Add 2 ml 1X Elution Buffer, Homogenize 5 min.

FRACTION 3: Centrifuge 700 g, 3 min., 4° C. Recover supernatant (2ml ).

Preserve an aliquot of some μl of each fraction, at 4° C., for itsmigration on 10% Acrylamide Gel.

Concentration and Elimination of Proteins of Small Molecular Weight

Pool the three fractions=4-5 ml and chromatography them on Ultra-4column AMICON, 50000NMWL, (Amicon Ultra centrifugal filter devices withlow-binding Ultracel membrane—MILLIPORE, Ref. UFC8 050 24).

Centrifuge 15 min., 3000 g, 25° C.

Washing and/or change buffer by addition to the column of 4 ml of 100 mMTris pH 8 buffer.

Centrifuge 15 min., 3000 g, 25° C.

Obtaining of 30 μl of each purified and concentrated protein.

Addition of 270 μl of storage buffer [50 mM Tris HCl pH8; 100 mM NaCl;0,1 mM EDTA; 1 mM DTT] (final volume=300 μl)

The concentrated and purified proteins are stored at 4° C.

The purity and the concentration of the purified polymerases areevaluated by SDS PAGE.

-   Protein dosage is carried out on 1/10 diluted samples in Tris 100 mM    pH 8 buffer, with the BioPhotometer 6131 (Eppendorf)

Purification Control

Polyacrylamide gels were stained with Coomassie Blue (see FIGS. 6 and7);

M is the SDS PAGE molecular weight standards. Low Range (BIO-RAD, Ref.161-0304). Bands at 97.4 kDa; 66.2 kDa; 45 kDa; 31 kDa; 21 kDa and 14.4kDa.

Catalytic Properties of the Polymerases Example 7 Primer Extension

The three variants of interest (proteins with the histidine tag) wereused in a primer extension assay using radiolabelled primers. Thesevariants have a strong DNA-dependent DNA-polymerase activity at 65° C.For two of them (b and a) this activity was almost the same as that ofthe fragment of Stoffel (e) produced and purified under the sameconditions (see FIG. 8: Gel 1).

On the other hand, reverse transcriptase activity detected at 65° C. forboth variants d and b (respectively a) is much higher than the reversetranscriptase activity obtained in the presence of magnesium for thefragment e (see FIG. 8: Gel 2).

The present inventors also checked the thennal stability of thesevariants. The DNA-dependent DNA-polymerase activity was maintained afteran incubation of proteins 45 min. at 65° C. This maintenance of thecatalytic activity DNA-dependent DNA-polymerase on variants wasnecessary for the design of a protocol of “RT-PCR one-pot”.

The inventors confirm that the method of the present invention shouldallow the acquisition of polymerase variants having the catalyticactivities required for such of “RT-PCR one-pot” protocol. Oneembodiment is given in Example 9.

Products of Primer Extension With Polymerases e, a, d and b

DNA polymerases e, a, b and d were prepared and further purified usingthe six-histidines tag as described in the paragraph before Example 6.

Materials

A mixture consisting of 1 μl of variant histidine tagged enzymes e (960mg/l), a (870 mg/l), b (910 mg/l) and d (510 mg/l), template (theoligoribonucleotide of SEQ ID NO: 12 or the deoxyribonucleotide of SEQID NO: 14), DNA primer 13 (SEQ ID NO: 13), 0.25 mM dNTP, 3 mM MgCl₂ inbuffer B 1× was incubated at 65° C. for 5 minutes. After denaturation at94° C., the samples were loaded on a 20% polyacrylamide gel forelectrophoresis.

Method

GEL 1: 1 μl of the concentrated fraction of each variant is added to 15μl of the reaction mixture [0.5 μM DNA template(AAATACAACAATAAAACGCCACATCTTGCG; SEQ ID NO 14); 0.5 μM DNA(TAACACGACAAAGCGCAAGATGTGGCGT; SEQ ID NO: 13); 0.25 mM dNTP; 3 mM MgCl₂;1X buffer C].

Polymerization is carried out at 65° C. during 5 min.

The reaction is stopped by addition of 15 μl of solution ofdenaturation, heated at 94° C. and then placed at 4° C.

15 μl of each sample are deposited onto the gel.

GEL 2: 1 μl of the concentrated fraction of each variant is added to 15μl of the reaction mixture [10 μM RNA (AAAUACAACAAUAAAACGCCACAUCUUGCG;SEQ ID NO: 12); 5 μM DNA (TAACACGACAAAGCGCAAGATGTGGCGT; SEQ ID NO: 13);0.25 mM dNTP; 3 mM MgCl₂, 1X buffer C].

Polymerization is carried out at 65° C. during 5 min.

The reaction is stopped by addition of 15 μl of solution ofdenaturation, heated at 94° C. and then is placed at 4° C.

15 μl of each sample are deposited on each gel.

-   The results for gels 1 and 2 are shown in FIG. 8.

Example 8 Optimization of PCR

The present inventors have developed a PCR protocol for theamplification of DNA fragments. The hybridization of the primers to thetemplate and the polymerization are carried out at 65° C. The PCRreaction of a DNA template by variants a and e were tested. Materialsand methods are described below and results are shown in FIG. 9.

Furthermore, the PCR reaction of a DNA template by variant a, for whichkinetic analysis was made, was tested on two different thermocyclers (MJResearch and Applied Biosystems), the results obtained with thesemachines are comparable for the amplification of a fragment of about 450bp (see FIG. 9). This result was extended to the amplification of afragment of about 1560 bp (see FIG. 10).

Briefly, the catalytic properties of variant a evaluated by PCR are verysimilar to those of the Stoffel fragment of the Taq polymerase (e).

-   Products of PCR with polymerase e and variant a (FIG. 9):

PCR Reaction Mixture (for 20 μl)

Variants a and e (purified and diluted 1/10 in Tris 100 mM) 1.5 μlThermophilic DNA Polymerase 10× 2 μl buffer, Magnesium Free, PromegaPrimer 1 fwd (100 μM) 0.125 μl Primer 2 rev (100 μM) 0.125 μl dNTP at 25mM 0.2 μl DNA template (pGL2-luciferase, 10 ng) 1 μl MgCl₂ (25 mM) 1 μlH₂O 9.05 ml

Temperature Cycle on PTC-100 (MJ RESEARCH)

94° C., 1 min.; [94° C., 20 sec; 65° C., 4 min]_(40 cycles); 65° C., 15min.

Sequences of the Primers

Primer 1 fwd: GGA TGG AAC CGC TGG AGA GCA ACT G (SEQ ID NO: 101) Primer2 rev: GAT CTC TCT GAT TTT TCT TGC GTC G (SEQ ID NO: 102)

In a 20 μl reaction volume, 1.5, μl of polymerases e (960 mg/l) or a(870 mg/l) were mixed with 10 ng of template (plasmid pGL2-luciferase,Promega), 0.6 μM primer 111 (GGA TGG AAC CGC TGG AGA GCA ACT G; SEQ IDNO: 101), 0.6 μM primer 112 (GAT CTC TCT GAT TTT TCT TGC GTC G; SEQ IDNO: 102)), 0.25 mM dNTP, 2.5 mM MgCl₂ in buffer B 1X prior to incubationat the following temperature/time steps (94° C./1 min.) (94° C./20 sec;65° C./4 min)₄₀ cycles (65° C./15 min.) on a PTC100 thermocycler (MJResearch) to yield an about 1560 bp long PCR product as shown on thefigure representing the agarose gel after electrophoresis. M is themarker Smartladder (Eurogentec).

Variant a: obtaining a fragment of 475 bp (FIG. 10)

Reaction mixture for PCR (for 20 μl of reaction)

Variant a 1.5 μl Thermophilic DNA Polymerase 10× 2 μl buffer, MagnesiumFree, Promega Primer 1 Fwd (50 μM) 0.5 μl Primer 2 Rev (50 μM) 0.5 μldNTP set (25 mM) 0.2 μl MgCl₂ (25 mM) 1 μl DNA template (pHEN1-Taq,about 10 ng) 1 μl H₂0 13.3 μl

Temperature cycles on PTC-100 (MJ RESEARCH)

94° C., 1 min.; [94° C., 20 s; 65° C., 4 min]35 cycles; 65° C., 15 min.

Temperature cycles on GeneAmp PCR System-9700 (Applied BioSystems)

94° C., 1 min.; [94° C., 20 s; 66, 5° C., 4 min]40 cycles; 65° C., 15min.

Sequences of the Primers

(SEQ ID NO: 16) Primer 1 fwd: GAG AAG ATC CTG CAG TAC CGG GAG C (SEQ IDNO: 4) Primer 2 rev: TTC ATT CTT GCT AGC TCC TGG GAG AGG C

RT-PCR

Subsequent to optimisation of the temperature cycles, the buffer and theadditives used, the present inventors realized a reaction of “RT-PCRone-pot” with variant a without addition of reagents after the beginningof the reaction (Example 9 and FIG. 11). This result was been reproducedfor two distinct buffers containing magnesium ions (without manganeseions) on two different thermocyclers with the same batch from RNA(rabbit globin mRNA, Sigma R1253) and the same batch of variant a.

The present inventors tested this RNA in a “RT-PCR one-pot” reactionusing the commercial kit of Applied Biosystems (Gene Amp AccuRT RNAPCR). The RNA is amplified in the form of a slightly visible band.

Example 9 RT-PCR “one pot” (FIG. 11)

Reaction mixture of RT-PCR “one-pot” (or 25 μl of reaction)

Variant a 1 μl ( column 5); 0.5 (column 4) Thermophilic DNA Polymerase10× 2.5 μl buffer, Magnesium Free, Promega Primer 1 fwd (50 μM) 0.25 μlPrimer 2 rev (50 μM) 0.25 μl dNTP set (25 mM) 0.25 μl MgCl₂ (25 mM) 0.75μl mRNA globin SIGMA (20 ng/μl) 0.25 μl Adjuvant 1 1 μl Adjuvant 2 0.4μl H₂O 18.35 μl

Cycle Temperature on PTC-100 (MJ RESEARCH)

94° C., 15 s.; 65° C., 45 min.; [94° C., 20 s; 65° C., 4min]_(32 cycles); 65° C., 15 min.

Sequences of the Primers

Primer 1 fwd: TTG GCC AGG AAC TTG TCC (SEQ ID NO: 18) Primer 2 rev: GACCAA CAT CAA GAC TGC C (SEQ ID NO: 17)

Amplification product=372 bp.

In a 25 μl reaction volume, 0.5 or 1 ml of purified and sixhistidines-tagged polymerase a (117 mg/l) were mixed with 5 ng oftemplate (rabbit globin messenger RNA, Sigma), 0.5 μM primer 113 (GACCAA CAT CAA GAC TGC C; SEQ ID NO: 17), 0.5 μM primer 114 (TTG GCC AGGAAC TTG TCC (SEQ ID NO: 18), 0.25 mM dNTP in a manganese-free buffer B1X containing 1.25 mM MgCl₂, 1 mg acetamide and 8 μM tetramethylammonium chloride, prior to incubation at the following temperature/timesteps (94° C./15 s; 65° C./45 min.)(94° C./30 s; 65° C./4min)_(32 cycles) (65° C., 15 min.) on a thermocycler (Biometra) to yieldan about 372 bp long PCR product as shown in FIG. 11 representing theagarose gel after electrophoresis. M is the marker of phage PhiX DNAdigested by the restriction enzyme HaeIII.

Example 10 Kinetic Parameters for Variant a

For evaluation of the kinetic parameters, polymerase a afterpurification by affinity chromatography for the histidine tag wascleaved at 23° C. by the protease thrombin for release of the tag andfurther purified on a heparin column (Pharmacia) prior tocharacterisation by SELDI TOF mass spectrometry. Remaining His-taggedpolymerases were finally removed by incubation with Co²⁺ resin (TalonMetal Affinity resin).

k_(cat) 0.1 s⁻¹ K_(m)(dNTP) 3.10⁻⁵ mol l⁻¹ k_(cat)/K_(m)(dNTP) 3.10³ l ·mol⁻¹ · s⁻¹for the RNA templates (AAG CCU ACG ACU CCG AAC UGA CCG UGC UAC CAA U;SEQ ID NO: 103), (AAG CCU ACU ACU CCG AAC UGA CCG UGC UAC CAA U; SEQ IDNO: 104), (AAG CCU ACA ACU CCG AAC UGA CCG UGC UAC CAA U; SEQ ID NO:105) and for the primer DNA (A TTG GTA GCA CGG TCA GTT CGG AGT; SEQ IDNO: 106) and for dNTPs (N=A, C or T).

The catalytic efficiency for RNA-dependent DNA-polymerisation measuredas k_(cat)/K_(m)(dNTP) is about ten fold higher for variant a thanvariant e.

Example 11 Identification of New Variants

96 clones were isolated starting from the population selected fromphage-polymerases after the sixth cycle from evolution directed towardsthe reverse transcriptase activity. 91 genes coding for the variant ofpolymerase were sequenced and characterized. 19 additional and distinctsequences to those described in the examples above, labelled “rtX”appearing in the table below, were identified. The correspondingphages-polymerases were prepared as previously described. The catalyticactivities of the phage-polymerases were controlled by primer extensionby using radio-labelled primers. In these assays for phage-polynierases,some variants have a whole DNA-dependent—DNA-polymerase activity at 65°C. In these assays, some variants seem to have a very strongRNA-dependent DNA-polymerase activity at 65° C. The results for thisstudy are shown in FIG. 12.

These results will enable the inventors to establish a link between thesequence and the catalytic activity of the phages-polymerases selected.These data support a utility in “RT-PCR one-pot”.

Summary of the Taq Sequence Variants Above

In the N-terminus of the purified proteins, the signal sequenceappearing in SEQ ID NOs: 19-38 polynucleotide sequence—odd nunmberedsequences, protein sequence—even numbered sequences) is not taken inaccount, i.e., the peptide having the sequence MASG₄CG₄ (SEQ ID NO: 39),which has been introduced upstream of the sequence SPKA (amino acids13-16 of SEQ ID NO: 26). The latter sequence corresponds to the Stoffelfragment beginning (S being the amino acid occupying the position number290 in the Taq polymerase sequence).

In the C-terminus of the purified proteins, the sequence AAALVPRGSLEH₆(SEQ ID NO: 40) comprising a site of cleavage by thrombin appearing inSEQ ID NOs: 19-38 (polynucleotide sequence—odd numbered sequences,protein sequence—even numbered sequences), as well as a polyhistidinetag that was introduced to facilitate further purification of theprotein are also not taken into account or are not essential for thesequences of the present invention.

Further, the C-terminal alanine dipeptide appearing in SEQ ID NOs: 62-99(polynucleotide sequence—even numbered sequences, protein sequence—oddnumbered sequences) can similarly be removed to obtain the sequences ofthe present invention.

Clearly, the present invention contemplates and embraces sequences thathave been modified to contain one or more of the following: a N-terminalleader/signal sequence, an N-terminal fusion, the remainder of theN-terminal region (residues 1-200) of the Taq polymerase wild-typesequence SEQ ID NO: 100, a C-terminal cap resulting from proteasecleavage (e.g., thrombin), a C-terminal fusion, a C-terminalpurification tag, etc.

In a particularly preferred embodiment of the preseint invention are thefollowing variants of the Taq polymerase. More particularly preferredare the variants represented by residues 13-555 of SEQ ID NOs: 20, 22,24, 28, 30, 32, 34, 36, and 38, and the variants represented by residues1-543 of SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, and 98.

Mutations assessment^(1,2) sequence SEQ ID NO: M761V “s” 38 M761T,D547G, I584V “a” 20 W827R “m” 32 W827R, E520G, A608T “b” 22 W827R,A517V, T664S, F769S “g” 28 M747K, Q698L, P816L “n” 34 M747R, W604R,S612N, V730L, R736Q, “d” 24 S739N, N483Q, S486Q, T539N, Y545Q, D547T,P548Q, A570Q, D578Q, A597T F749Y, A568V “i” 30 F749Y, P550Q, R556S,V740E, V819A “q” 36 Stoffel Fragment (e)³ “e” 26 E530G, Y696C, A803V“rt1” 63 H480R, W827R “rt2” 65 V586I, I638F, M747K, G776E “rt3” 67T509S, L552H, M779K “rt16” 69 Q534R, A764T “rt18” 71 F482L, T664A,F749Y, P770T, M775T “rt25” 73 L552P, A661T, A800V, E820K “rt26” 75I553F, A608V, F749S “rt28” 77 E742K, M747K “rt30” 79 M747R “rt31” 81A608V “rt33” 83 R651Q “rt36” 85 M747K “rt43” 87 Y696N “rt59” 89 Y696C“rt64” 91 I599N, L780P, E820K “rt70” 93 Y696C, K767E “rt78” 95D551G,V783I “rt80” 97 S612N, P816S “rt86” 99 ¹Amino acid numberscorrespond to position in the Taq polymerase wild-type sequence SEQ IDNO: 100. ²All sequences contain a R795G mutation (i.e., residuecorresponding to 518 in the sequence of SEQ ID NO: 26); however, thismutation need not be present within the context of the presentinvention. In other words, the present invention embraces variantscorresponding to the above in which R795 is conserved. ³Wild-typeStoffel fragment (residues 13-555 of SEQ ID NO: 26) listed for referencepurposes; however, as stated herein above, variant e also contains aR795G mutation, which is not reflected in SEQ ID NO: 26 appearing in theSequence Listing.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

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1. A purified polynucleotide which encodes a thermostable polypeptidecomprising an amino acid sequence having at least 80% identity toresidues 13-555 of SEQ ID NO: 26, wherein said polypeptide has at leastone mutation in amino acids 461 to 490 of SEQ ID NO: 26, or at aposition selected from the group consisting of H203, F205, T232, E253,Q257, D274, L275, 1276, V309, 1322, A331, L332, D333, Y334, S335, I361,R374, A384, T387, Y419, P493, M498, G499, M502, L503, V506, R518, A523,A526, P539, E543, and W550, and wherein said polypeptide has DNApolyrerase activity.
 2. The purified polynucleotide of claim 1, whereinsaid at least one mutation is selected from the group consisting ofA331T, S335N, M470K, M470R, F472Y, M484V, M484T, R518, and W550R.
 3. Thepurified polynucleotide of claim 1, wherein said polypeptide has atleast 90% identity to residues 13-555 of SEQ ID NO:
 26. 4. The purifiedpolynucleotide of claim 1, wherein said polypeptide has at least 95%identity to residues 13-555 of SEQ ID NO:
 26. 5. The purifiedpolynucleotide of claim 1, wherein said polypeptide has at least 97.5%identity to residues 13-555 of SEQ ID NO:
 26. 6. The purifiedpolynucleotide of claim 1, wherein said polypeptide comprises at leasttwo mutations.
 7. The purified polynucleotide of claim 1, wherein saidpolypeptide comprises an amino acid sequence corresponding to residues13-555 of a sequence selected from the group consisting of SEQ ED NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO:
 38. 8. The purifiedpolynucleotide of claim 1, wherein said polypeptide has a mutation atR518.
 9. The purified polynucleotide of claim 8, wherein said mutationat R518 is R518G.
 10. The purified polynucleotide of claim 1, whereinsaid at least one mutation is selected from the group consisting ofH203R, F205L, T232S, E253G, Q257R, D274G, L275H, L275P, I276F, V309I,I322N, A331V, S335N, I361F, R374Q, A384T, T387A, Y419C, Y419N, E465K,M470K, M470R, F472Y, F472S, A487T, K490E, P493T, M498T, G499E, M502K,L503P, V506I, R518G, A523V, A526V, P539S, E543K, and W550R.
 11. Thepurified polynucleotide of claim 10, wherein said polypeptide comprisesan amino acid sequence corresponding to residues 1-543 of a sequenceselected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 65, SEQID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ IDNO: 95, SEQ ID NO: 97, and SEQ ID NO:
 99. 12. The purifiedpolynucleotide of claim 1, wherein said polynucleotide comprises asequence corresponding to nucleotides 39-1667 of a sequence selectedfrom the group consisting of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, and SEQ ID NO:
 37. 13. The purified polynucleotide of claim 1,wherein said polynucleotide comprises a sequence corresponding tonucleotides 1-1629 of a sequence selected from the group consisting ofSEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ IDNO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, and SEQ ID NO:98.
 14. A purified polynucleotide that is complementary to thepolynucleotide of claim
 1. 15. A purified polynucleotide whichhybridizes under stringent conditions to the polynucleotide of claim 1,wherein said stringent conditions comprise washing in 5×SSC at atemperature from 50° C. to 68° C.
 16. A vector comprising the purifiedpolynucleotide of claim
 1. 17. The vector of claim 16, wherein saidpolynucleotide is operably linked to a heterologous expression sequence.18. A host cell comprising the purified polynucleotide of claim
 1. 19. Apurified thermostable polypeptide comprising an amino acid sequencehaving at least 80% identity to residues 13-555 of SEQ ID NO: 26,wherein said polypeptide has at least one mutation in amino acids 461 to490 of SEQ ID NO: 26, or at a position selected from the groupconsisting of H203, F205, T232, E253, Q257, D274, L275, I276, V309,I322, A331, L332, D333, Y334, S335, I361, R374, A384, T387, Y419, P493,M498, G499, M502, L503, V506, R518, A523, A526, P539, E543, and W550,and wherein said polypeptide has DNA polymerase activity.
 20. Thepurified polypeptide of claim 19, wherein said at least one mutation isselected from the group consisting of A331T, S335N, M470K, M470R, F472Y,M484V, M484T, R518G, and W550R.
 21. The purified polypeptide of claim19, wherein said polypeptide has at least 90% identity to residues13-555 of SEQ ID NO:
 26. 22. The purified polypeptide of claim 19,wherein said polypeptide has at least 95% identity to residues 13-555 ofSEQ ID NO:
 26. 23. The purified polypeptide of claim 19, wherein saidpolypeptide has at least 97.5% identity to residues 13-555 of SEQ ID NO:26.
 24. The purified polypeptide of claim 19, wherein said polypeptidecomprises at least two mutations.
 25. The purified polypeptide of claim19, wherein said polypeptide comprises an amino acid sequencecorresponding to residues 13-555 of a sequence selected from the groupconsisting of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQID NO:
 38. 26. The purified polypeptide of claim 19, wherein saidpolypeptide has a mutation at R518.
 27. The purified polypeptide ofclaim 26, wherein said mutation at R518 is R518G.
 28. The purifiedpolypeptide of claim 19, wherein said at least one mutation is selectedfrom the group consisting of H203R, F205L, T232S , E253G , Q257R , D274G, L275H , L275P, I276F, V309I , I322N , A331V , S335N, I361F, R374Q,A384T , T387A, Y419C, Y419N, E465K, M470K, M470R, F472Y, F472S, A487T,K490E, P493T, M498T, G499E, M502K, L503P, V506I, R518G, A523V, A526V,P539S, E543K, and W550R.
 29. The purified polypeptide of claim 28,wherein said polypeptide comprises an amino acid sequence correspondingto residues 1-543 of a sequence selected from the group consisting ofSEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ IDNO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ED NO: 97, and SEQ ID NO:99.
 30. A kit for amplifying DNA comprising: a purified thermostablepolypeptide, wherein said polypeptide has at least 80% homology toresidues 13-555 of SEQ ID NO: 26, wherein said polypeptide has at leastone mutation in amino acids 461 to 490 of SEQ ID NO: 26, or at aposition selected from the group consisting of H203, F205, T232, E253,Q257, D274, L275, I276, V309, I322, A331, L332, D333, Y334, S335, I361,R374, A384, T387, Y419, P493, M498, G499, M502, L503, V506, R518, A523,A526, P539, E543, and W550, and wherein said polypeptide has DNApolymerase activity; a concentrated buffer solution, and optionally oneor more divalent metal ions; and a mixture of deoxyribonucleotides. 31.The kit of claim 30, wherein said at least one mutation is selected fromthe group consisting of A331T, S335N, M470K, M470R, F472Y, M484V, M484T,R518G, and W550R.
 32. The kit of claim 30, wherein said divalent metalion is Mg²⁺ or Mn²⁺.
 33. The kit of claim 30, wherein said polypeptidehas at least 90% identity to residues 13-555 of SEQ ID NO:
 26. 34. Thekit of claim 30, wherein said polypeptide has at least 95% identity toresidues 13-555 of SEQ ID NO:
 26. 35. The kit of claim 30, wherein saidpolypeptide has at least 97.5% identity to residues 13-555 of SEQ ID NO:26.
 36. The kit of claim 30, wherein said polypeptide comprises at leasttwo mutations.
 37. The kit of claim 30, wherein said polypeptidecomprises an amino acid sequence corresponding to residues 13-555 of asequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, and SEQ ID NO:
 38. 38. The kit of claim 30,wherein said polypeptide has a mutation at R518.
 39. The kit of claim38, wherein said mutation at R518 is R51G.
 40. The kit of claim 30,wherein said at least one mutation is selected from the group consistingof H203R, F205L, T232S, E253G, Q257R, D274G, L275H, L275P, I276F, V309I,I322N, A331V, S335N, I361F, R374Q , A384T , T387A, Y419C, Y419N, E465K,M470K, M470R, F472Y, F472S, A487T, K490E, P493T, M498T, G499E, M502K,L503P, V506I, R518G, A523V, A526V, P539S, E543K, and W550R.
 41. The kitof claim 40, wherein said polypeptide comprises an amino acid sequencecorresponding to residues 1-543 of a sequence selected from the groupconsisting of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQIDNO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,and SEQ ID NO:
 99. 42. The kit of claim 30, further comprising a 5′ to3′ exonuclease or a 3′ to 5′ exonuclease.
 43. The kit of claim 42,wherein said 5′ to 3′ exonuclease has a sequence of SEQ ID NO:
 50. 44.The kit of claim 42, wherein said 3′ to 5′ exonuclease has a sequence ofSEQID NO:
 51. 45. A method for reverse transcribing an RNA comprising:a) providing a reverse transcription reaction mixture comprising saidRNA, a primer, a divalent cation, and a purified therrnostablepolypeptide comprising an amino acid sequence having at least 80%identity to residues 13-555 of SEQ ID NO: 26, wherein said polypeptidehas at least one mutation in amino acids 461 to 490 of SEQ ID NO: 26, orat a position selected from the group consisting of H203, F205, T232,E253, Q257, D274, L275, I276, V309, I322, A331, L332, D333, Y334, S335,I361, R374, A384, T387, Y419, M470, F472, M484, P493, M498, G499, M502,L503, V506, R518, A523, A526, P539, E543, and W550, and wherein saidpolypeptide has DNA polymerase activity; and b) treating said reactionmixture at a temperature and under conditions suitable for said purifiedpolypeptide to initiate synthesis of an extension product of said primerto provide a cDNA molecule complementary to said RNA.
 46. The method ofclaim 45, wherein said at least one mutation is selected from the groupconsisting of A331T, S335N, M470K, M470R, F472Y, M484V, M484T, R518G,and W550R.
 47. The method of claim 45, wherein said at least onemutation is selected from the group consisting of H203R, F205L, T232S,E253G, Q257R, D274G, L275H , L275P, I276F, V309I, I322N, A331V, S335N,I361F, R374Q, A384T, T387A, Y419C, Y419N, E465K, M470K, M470R, F472Y,F472S, A487T, K490E, P493T, M498T, G499E, M502K, L503P, V506I, R518G,A523V, A526V, P539S, E543K, and W550R.
 48. A method of identifyingthermnostable mutant polypeptides comprising: a) packaging a vector inwhich a polynucleotide encoding a phage coat protein is fused to apolynucleotide encoding a protein having at least 80% identity toresidues 13-555 of SEQ ID NO: 26 into a phage; b) expressing the fusionprotein; c) isolation of phage particles; d) infecting E. coli andincubating the infected E. coli; e) detecting the fusion protein; and f)assessing polymerase activity.
 49. The method of claim 48, wherein b) tof) are repeated 0 to 25 times.
 50. The method of claim 48, wherein thephage coat protein is fused to said polynucleotide encoding a proteinhaving at least 80% identity to residues 13-555 of SEQ ID NO: 26 by wayof a linker having a sequence represented by residues 3-12 of SEQ ID 39.51. A method of identifying thermnostable mutant polypeptides having acatalytic activity comprising: a) packaging a vector in which a gene orfragment thereof encoding variants of a catalytic domain responsible forthe catalytic activity fused to a gene encoding a phage coat protein; b)isolation and purification of phage particles; c) heating thephage-mutant polypeptide at a temperature ranging from 50° C. to 90° C.for a time ranging from 30 seconds to several hours; d) cross-linking aspecific substrate with a phage particle; e) forming a reaction productfrom the substrate catalyzed by the thermostable mutant protein onphage, wherein the temperature is optionally regulated to be the same orgreater or lower than the temperature of c); f) selecting the phageparticles comprising a variant nucleotide sequence encoding for thecatalytic domain responsible for the catalytic activity at the regulatedtemperature, by capturing the reaction product or screening for saidreaction product; g) infecting E. coli with the phage particles selectedat f); h) incubating the infected E. coli; and i) assessing catalyticactivity of the proteins corresponding to isolated genes.
 52. The methodof claim 51, wherein the gene or fragment thereof encoding variants of acatalytic domain is directly fused to the gene encoding a phage coatprotein.
 53. The method of claim 51, wherein a) to h) are repeated 0 to20 times.
 54. The method of claim 51, wherein the gene or fragmentthereof encoding variants of a catalytic domain and the gene encoding aphage coat protein, are indirectly fused by a peptide or polypeptidelinker.
 55. The method of claim 54, wherein the peptide is selected fromthe group consisting of: a glycine rich linker represented by(SG₄)_(n)(SEQ ID NO: 107): a glycine rich linker represented by SG₄CG₄(residues 3-12 of SEQ ID NO: 39); a human calmodulin (SEQ ID NO: 46);and a hexahistidine (SEQ ID NO: 108) binding single chain variablefragment consisting of: (i) an anti-His Tag Antibody 3D5 Variable HeavyChain (SEQ ID NO: 47); (ii) a linker (SEQ ID NO: 48); and (iii) ananti-His Tag Antibody 3D5 Variable Light Chain (SEQ ID NO: 49).
 56. Themethod of claim 54, wherein the polyp eptide linker is selected from thegroup consisting of: a protein binding the substrate at hightemperature; a catalytic domain of a 5′ to 3′ exonuclease; a catalyticdomain of a 3′ to 5′ exonuclease; a catalytic domain of Bacilluscirculans cyclodextringlycosyltransferase (SEQ ID NO: 52); a catalyticdomain of Bordetella pertussis adenylate cyclase (SEQ ID NO: 53); aBacillus amyloliquefaciens serine protease subtilisin (SEQ ID NO: 54);and a catalytic domain of Bacillus subtilis lipase A (SEQ ID NO: 55).57. The method of claim 51, wherein the cross-linking between thespecific substrate of the catalytic domain of the polyp eptide with thephage particle is made by a cross-linking agent selected from the groupconsisting of: a maleimidyl group; an iodoacetyl group disulfide andderivative; and any other thermostable link.
 58. The method of claim 51,wherein the catalytic domain is the catalytic domain of an enzymeselected from the group consisting of: a DNA polymerase; analpha-amylase; a lipase; a protease; a cyclodextringlycosyltransferase;and an adenylate cyclase.
 59. The method of claim 51, wherein theassessment of the catalytic activity of f) is made by means of a DNApolymerization.
 60. The method of claim 51, wherein b) is performedafter e) or during h).
 61. The method of claim 51, wherein thetemperature in e) is regulated to be the same or greater than thetemperature of c).
 62. The method of claim 51, wherein the temperaturein e) is regulated to be the same or less than the temperature of c).63. A method of obtaining a thermostable variant enzyme comprising: a)screening enzymes expressed at the surface of phage particles andidentifying at least a thermostable variant conserving its activecatalytic domain at a regulated temperature according to the method ofclaim 62; b) isolating and sequencing a DNA encoding said identifiedthermostable variant; c) preparing a vector comprising the DNA of b); d)transfecting or infecting cells with the vector obtained at c); e)expressing the thermostable variant enzyme from the cells andoptionally; and f) recovering, isolating and purifying said thermostablevariant enzyme expressed at e).
 64. A method of obtaining a thermostablevariant enzyme comprising: a) screening enzymes expressed at the surfaceof phage particles and identifying at least a thermostable variantconserving its active catalytic domain at a regulated temperatureaccording to the method of claim 61; b) isolating and sequencing a DNAencoding said identified thermostable variant; c) preparing a vectorcomprising the DNA of b; d) transfecting or infecting cells with thevector obtained at c); e) expressing the thermostable variant enzymefrom the cells and optionally; and f) recovering, isolating andpurifying said thermostable variant enzyme expressed at e).
 65. Aninsert contained in a phage selected from the group consisting ofI-3168, I-3169, I-3170, I-3171, I-3172, I-3173, I-3174, I-3175, I-3176and I-3158 in CNCM on Feb. 27,
 2004. 66. A recombinant host cellcomprising an insert or a polynucleotide encoding a thermostablepolymnerase according claim 65.